The Role of Dna Methylation In Addiction And

THE ROLE OF DNA METHYLATION
IN ADDICTION AND DEPRESSION
APPROVED BY SUPERVISORY COMMITTEE
Dr. Eric J. Nestler, M.D., Ph.D.
Dr. Christopher Cowan Ph.D.
Dr. Craig Powell, M.D., Ph.D.
Dr. Joel Elmquist, D.V.M., Ph.D.
DEDICATION
I’d first and foremost like to thank my parents and siblings for their unconditional love
and support. I’d also like to thank Dr.’s He Liu and Harold Zakon at the University of
Texas at Austin for originally (and patiently) introducing me into basic research in
neuroscience. Similarly, I’d like to thank Dr.’s Nancy Street, Jay Gibson, and Scott
Cameron for their mentorship in the SURF (Summer Undergraduate Research
Fellowship) program at UT Southwestern—these experiences in the lab were
instrumental in guiding me to a career as a physician-scientist.
Many thanks to PhD mentor, Dr. Eric J. Nestler, for his guidance and support throughout
the highs and lows of my graduate years. He has created an amazing environment
(empire) to work in and is an incredible role model. I would also like to thank Dr. Vincent
Vialou and Dr. Herbert E. Covington III. In addition to their friendship, Dr.’s Vialou and
Covington were both instrumental in introducing me to surgical and behavioral
techniques commonly used in the depression and addiction fields. Also, thanks to all
other members (present and past) in the Nestler lab—it’s been great working with you
all.
Thanks to all the members of my thesis committee: Dr. Christopher Cowan, Dr. Joel
Elmquist, Dr. Craig Powell for your assistance, constructive input, and criticism during
my graduate career.
Special thanks to my girlfriend Dani Dumitrui, and her daughters, Alexa and Nikita. This
is truly the best time of my life, and I have you to thank for it. Thanks for being patient
with me while working on this dissertation.
Finally, I would like to dedicate this thesis to the people that have struggled from or are
currently struggling with depression or addiction. I hope that this work will become a
small step towards elucidating the underpinnings of these terrible diseases.
THE ROLE OF DNA METHYLATION
IN ADDICTION AND DEPRESSION
by
QUINCEY CHARLES LAPLANT
DISSERTATION
Presented to the Faculty of the Graduate School of Biomedical Sciences
The University of Texas Southwestern Medical Center at Dallas
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
The University of Texas Southwestern Medical Center at Dallas
Dallas, Texas
June, 2010
THE ROLE OF DNA METHYLATION
IN ADDICTION AND DEPRESSION
QUINCEY CHARLES LAPLANT, M.D., Ph.D.
The University of Texas Southwestern Medical Center at Dallas, 2010
ERIC J. NESTLER, M.D., Ph.D.
Despite abundant expression of DNA methyltransferases (Dnmt’s) in brain, the regulation and
behavioral role of DNA methylation remain poorly understood. I found that Dnmt3a expression is
persistently regulated in nucleus accumbens (NAc), a key brain reward region, by chronic cocaine
or chronic social defeat stress. Moreover, NAc specific manipulations that block DNA
methylation potentiate cocaine reward and exert antidepressant-like effects, whereas NAc specific
Dnmt3a overexpression attenuates cocaine reward and is pro-depressant. On a cellular level, I
discovered that chronic cocaine selectively increases thin dendritic spines on NAc neurons and
that DNA methylation is both necessary and sufficient to mediate these effects. I then used
complementary genome wide techniques aimed at identifying cocaine-induced DNA methylation
at gene targets. Transcriptional profiling experiments in which I virally overexpressed Dnmt3a or
pharmacologically blocked DNA methylation identified an important role of DNA methylation in
iv
regulating the mRNA expression of a number of immediate early genes—several of which are
known to regulate dendritic spine morphology. Taken together, these data establish the
importance of Dnmt3a in the NAc in regulating molecular, cellular, and behavioral plasticity to
emotional stimuli.
v
Table of contents
DEDICATION …………………………….………………………………………….…………..II
ABSTRACT …………………………….……………………………………………………….IV
TABLE OF CONTENTS …………….………………………………………………….………VI
LIST OF FIGURES …….………...……...……………………………………………………...VII
LIST OF TABLES ………………...…………………………………………..……………….VIII
LIST OF ABBREVIATIONS …...……………………………………………………….………IX
PRIOR PUBLICATIONS ……………..…………………………………..……………………..XI
CHAPTER 1 – Introduction …………………………………………………………...…………..1
CHAPTER 2 – Dnmt3a regulates emotional behavior and spine plasticity in the nucleus
accumbens…………………………………………………………………………….…..............37
CHAPTER 3 – Genome wide analysis of cocaine-induced DNA methylation…………………..81
CHAPTER 4 –Conclusion………………………………………………………………………120
BIBLIOGRAPHY ……………………………………………………………………...……….129
vi
LIST OF FIGURES
CHAPTER 1 FIGURES
FIGURE 1-1…………………………………………………………………………………………31
FIGURE 1-2…………………………………………………………………………………………34
CHAPTER 2 FIGURES
FIGURE 2-1…………………………………………………………………………………………64
FIGURE 2-2…………………………………………………………………………………………66
FIGURE 2-3…………………………………………………………………………………………68
FIGURE 2-4…………………………………………………………………………………………70
FIGURE 2-5…………………………………………………………………………………………72
CHAPTER 2 SUPPLEMENTAL FIGURES
FIGURE 2-S1…………………………………………………………………………..……………74
FIGURE 2-S2…………………………………………………………………………………..……76
FIGURE 2-S3…………………………………………………………………………..……………77
FIGURE 2-S4…………………………………………………………………………..……………79
CHAPTER 3 FIGURES
FIGURE 3-1...……………………………………………………………………………………...106
FIGURE 3-2...……………………………………………………………………………………...108
FIGURE 3-3...……………………………………………………………………………………...110
FIGURE 3-4...……………………………………………………………………………………...112
FIGURE 3-5...……………………………………………………………………………………...114
CHAPTER 3 SUPPLEMENTAL FIGURES
FIGURE 3-S1...….………………………………………………………………………….……...119
vii
LIST OF TABLES
CHAPTER 1 TABLES
TABLE 1-1…………………………………………………………………………………………28
TABLE 1-2…………………………………………………………………………………………29
CHAPTER 2 TABLES
TABLE 2-1…………………………………………………………………………………………80
CHAPTER 3 TABLES
TABLE 3-1...……………………………………………………………………………………...116
TABLE 3-2...……………………………………………………………………………………...118
viii
LIST OF ABBREVIATIONS
∆FosB
AAV
acH3,acH4
AMPA
ARC
ATF1
ATF2
BDNF
bp
CamKII
CART
CBP
CDK5
c-Fos
ChIP
ChIP-chip
ChIP-Seq
CREB
DARPP-32
DNA
DNMT
Dnmt1, 3a, 3b
EGR
FosB
G9a
H2A,H2B,H3,H4
H3K27
H3K4
H3K9
H3K9me2
H3pS10
H3S10
HAT
HDAC
HDAC 1, 2, 4, 5, 7, 9
HMT
HSV
IEG’s
LTD
LTP
Truncated form of FosB that is induced by chronic drug use
Adeno-associated virus
Acetylated histone H3 or H4
Glutamate receptor (AMPA)
Activity-regulated cytoskeleton-associated protein
Activating transcription factor 1
Activating transcription factor 2
Brain derived neurotrophic factor
Base pair
Calcium/calmodulin-dependent protein kinase II
Cocaine and amphetamine regulated transcript
CREB-binding protein
Cyclin-dependent kinase 5
FBJ murine osteosarcoma viral oncogene
Chromatin immunoprecipitation
ChIP combined with microarrays
ChIP combined with massively parallel sequencing
cAMP response element binding protein
dopamine and cAMP regulated phosphoprotein of 32 kDa, DARPP-32
Deoxyribonucleic acid
DNA methyltransferase
DNA methyltransferase 1, 3a, 3b
Early growth response protein
FBJ murine osteosarcoma viral oncogene homolog B
KMT1c
Histone H2A, H2B, H3, H4
Histone H3 methylated at lysine 27 (repressive)
Histone H3 methylated at lysine 4 (activating)
Histone H3 methylated at lysine 9 (repressive)
dimethylation of histone H3 at Lys9
phosphorylation of histone H3 at Ser10
Histone H3 phosphorylated at serine 10 (activating)
Histone acetyltransferase
Histone deacetylase
Histone deacetylase 1, 2, 4, 5, 7, 9
Histone methyltransferase
Herpes simplex virus
immediate early genes
long term depression
long term potentiation
ix
MBD
mDIP
meDNA
MEF2
mEPSC
MS-275
MSK1
MSN
NAc
Nf-κB
NMDA
NPY
pacH3
PCR
PP1
PTMs
RG108
RNA
SAHA
SAM
SIRT1,2
TSA
TSS
Methyl-CpG-binding domain protein
methylated DNA immunoprecipitation
methylated DNA
Myocyte enhancer factor 2
miniature excitatory post-synaptic current
HDAC inhibitor
mitogen- and stress-activated kinase 1
Medium spiney neurons
Nucleus accumbens
Nuclear factor kappaB
N-methyl-D-aspartate
Neuropeptide Y
phosphoacetylated histone H3
Polymerase chain reaction
Protein phosphatase 1
post translational modification
DNA methyltransferase inhibitor
Ribonucleic acid
HDAC inhibitor (suberoylanilide hydroxamic acid)
S-adenosyl-L-methionine
Sirtuin 1,2
Trichostatin A (HDAC inhibitor)
transcription start site
x
PUBLICATIONS FROM MY THESIS WORK
1.
LaPlant Q, Vialou V, Covington HE 3rd, Dumitriu D, Feng J, Warren B, Maze I,
Dietz DM, Watts EL, Iñiguez SD, Koo JW, Mouzon E, Renthal W, Hollis F,
Wang H, Noonan MA, Ren Y, Eisch AJ, Bolaños CA, Kabbaj M, Xiao G, Neve
RL, Hurd YL, Oosting RS, Fan G, Morrison JH, Nestler EJ “Dnmt3a regulates
emotional behavior and spine plasticity in the nucleus accumbens.” Nature
Neuroscience 2010 (in review)
2.
Vialou V*, Maze I*, Renthal W, LaPlant Q, Watts EL, Mouzon E, Ghose S, Ginty
D, Tamminga C, Nestler EJ. “Serum response factor promotes resilience to chronic
social stress through the induction of ΔFosB” The Journal of neuroscience 2010 (in
review)
3.
Covington HE 3rd, Lobo MK, Vialou V, Maze I, Hyman J, LaPlant Q, Mouzon E,
Ghose S, Tamminga C, Neve RL, Deisseroth K, Nestler EJ. “Antidepressant effect of
optogenetic stimulation of the medial prefrontal cortex” The Journal of neuroscience.
2010 (in review)
4.
Vialou V, Robison AJ*, Laplant Q*, Covington HE 3rd, Dietz DM, Ohnishi YN,
Mouzon E, Rush AJ 3rd, Watts EL, Wallace DL, Iñiguez SD, Ohnishi YH,
Steiner MA, Warren BL, Krishnan V, Bolaños CA, Neve RL, Ghose S, Berton O,
Tamminga CA, Nestler EJ. “ΔFosB in brain reward circuits mediates resilience to
stress and antidepressant responses” Nature Neuroscience 2010 May 16. [Epub
ahead of print]
5.
Maze I, Covington HE 3rd, Dietz DM, LaPlant Q, Renthal W, Russo SJ,
Mechanic M, Mouzon E, Neve RL, Haggarty SJ, Ren Y, Sampath SC, Hurd YL,
Greengard P, Tarakhovsky A, Schaefer A, Nestler EJ. ”Essential role of the
histone methyltransferase G9a in cocaine-induced plasticity.” Science. 2010 Jan
8;327(5962):213-6.
6.
Covington HE 3rd, Maze I, LaPlant Q, Vialou VF, Ohnishi YN, Berton O, Fass
DM, Renthal W, Rush AJ 3rd, Wu EY, Ghose S, Krishnan V, Russo SJ,
Tamminga C, Haggarty SJ, Nestler EJ. “Antidepressant actions of histone
deacetylase inhibitors.” The Journal of neuroscience. 2009 Sep 16;29(37):1145160.
7.
Wilkinson MB*, Xiao G*, Kumar A*, LaPlant Q, Renthal W, Sikder D, Ghose
S, Tamminga C, Kodadek T, Nestler EJ (2009) “Imipramine treatment and
resiliency exhibit similar chromatin regulation in the mouse nucleus accumbens in
depression models.” The Journal of neuroscience. 29(24):7820-32.
8.
Renthal W*, Kumar A*, Xio G*, Wilkinson M, Covington HE 3rd, Maze I,
Sikder D, Robison AJ, LaPlant Q, Dietz DM, Russo SJ, Vialou V, Chakravarty
S, Kodadek TJ, Stack A, Kabbaj M, Nestler EJ. (2009) “Genome-wide analysis of
xi
chromatin regulation by cocaine reveals a role for sirtuins.” Neuron. 62(3):33548.
9.
LaPlant Q*, Chakravarty S*, Vialou V, Mukherjee S, Koo JW, Kalahasti G,
Bradbury KR, Taylor SV, Maze I, Kumar A, Graham A, Birnbaum SG, Krishnan
V, Truong HT, Neve RL, Nestler EJ, Russo SJ. (2009) Role of nuclear factor
kappaB in ovarian hormone-mediated stress hypersensitivity in female mice. Biol
Psychiatry. 15;65(10):874-80.
10.
Winstanley CA, Green TA, Theobald DE, Renthal W, LaPlant Q, Dileone RJ,
Chakravarty S, Nestler EJ. (2008) “DeltaFosB induction in orbitofrontal cortex
potentiates locomotor sensitization despite attenuating the cognitive dysfunction
caused by cocaine.” Pharmacol Biochem Behav.
11.
Zachariou V, Liu R, LaPlant Q, Xiao G, Renthal W, Chan GC, Storm DR,
Aghajanian G, Nestler EJ. (2008) “Distinct roles of adenylyl cyclases 1 and 8 in
opiate dependence: behavioral, electrophysiological, and molecular studies.” Biol
Psychiatry. 63(11):1013-21.
12.
Krishnan V*, Han MH*, Graham DL, Berton O, Renthal W, Russo SJ, LaPlant
Q, Graham A, Lutter M, Lagace DC, Ghose S, Reister R, Tannous P, Green TA,
Neve RL, Chakravarty S, Kumar A, Eisch AJ, Self DW, Lee FS, Tamminga CA,
Cooper DC, Gershenfeld HK, Nestler EJ. (2007). "Molecular adaptations
underlying susceptibility and resistance to social defeat in brain reward regions."
Cell 131(2): 391-404.
13.
Winstanley CA, LaPlant Q, Theobald DE, Green TA, Bachtell RK, Perrotti LI,
DiLeone RJ, Russo SJ, Garth WJ, Self DW, Nestler EJ. (2007) "DeltaFosB
induction in orbitofrontal cortex mediates tolerance to cocaine-induced cognitive
dysfunction." The Journal of neuroscience 27(39): 10497-507.
14.
Kumar A, Choi KH, Renthal W, Tsankova NM, Theobald DE, Truong HT, Russo
SJ, LaPlant Q, Sasaki TS, Whistler KN, Neve RL, Self DW, Nestler EJ. (2005).
"Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity
in striatum." Neuron 48(2): 303-14.
15.
Shah S, Lee SF, Tabuchi K, Hao YH, Yu C, LaPlant Q, Ball H, Dann CE 3rd,
Südhof T, Yu G. (2005) "Nicastrin functions as a gamma-secretase-substrate
receptor." Cell 122(3): 435-47.
Invited Review
1.
LaPlant Q, Nestler EJ. “CRACKing the histone code: Cocaine’s effects on
chromatin structure and function.” Hormones and Behavior (In Press).
xii
1
CHAPTER 1
INTRODUCTION
Preamble – The neuroplasticity paradox (why study epigenetics?)
Essentially all known mechanisms of long-lived neuronal plasticity involve
transient intracellular signaling cascades. And over the past several decades
neuroscientists have unraveled many mysteries of how these signaling events
translate into persistent changes in synaptic function (changes which may
underlie the basis of learning and memory). On a molecular scale, key examples
include insertion/removal of receptor subunits from synaptic membranes,
alteration of synaptic protein function via posttranslational modifications, and the
regulation of the translation or degradation of proteins at the synapse. However,
despite the “nobel” worthiness of these findings, a paradox exists. Despite the
persistence of many neuronal adaptations, all the above mechanisms occur at
the protein level and are therefore subject to eventual turnover. Neuroscientists
have long ignored the fact that the only permanent structure in any given neuron
is its genome. Therefore, these transient intracellular signaling cascades that
initiate all forms of neuronal plasticity must in some way be recorded in the
genome. In order to adapt properly (and persistently), each neuron must retain a
“memory” of where it has been—what genes were expressed, and just as
importantly what genes were not.
Thanks to advances in sequencing and
advances in our understanding of epigenetics, efforts are underway to identify
the missing link in the neuroplasiticy paradox. Is it DNA methylation?
2
Drug addiction introduction
Drug addiction is a debilitating psychiatric disorder characterized by
compulsive drug seeking and taking despite severe adverse consequences
(Hyman et al., 2006; Kalivas et al., 2005; Koob and Kreek, 2007). Once a person
succumbs to addiction, few effective therapies exist. Even when former addicts
remain abstinent for long durations of time, they often find themselves in a
lifelong struggle—always vulnerable to drug relapse. Therefore, the two major
questions on which basic science research focuses relate to understanding the
molecular events that occur during: 1) the transition to the addicted state and 2)
the maintenance of the addicted state. A better understanding of these
mechanisms would provide insight into how we can block or perhaps reverse the
neuroplastic changes that define addiction.
Drug-induced changes in gene expression in key brain reward regions,
such as the nucleus accumbens (NAc), prefrontal cortex (PFC), and ventral
tegmental area (VTA), represent one mechanism thought to contribute to both of
these key questions. A multitude of microarray studies under different
experimental conditions have found drug-induced alterations in the expression
levels of hundreds of mRNAs in these target regions (Freeman et al., 2010;
Heiman et al., 2008; Maze et al., 2010; McClung and Nestler, 2003; McClung et
al., 2005; Renthal et al., 2007; Winstanley et al., 2007; Yao et al., 2004). In
response to psychostimulants, many genes, such as those encoding c-Fos,
FosB, ΔFosB, ATF2 (activating transcription factor 2), ATF3, and ATF4, are
3
rapidly and transiently induced in response to initial drug exposures. Chronic
exposure differentially affects the steady state levels of these various mRNA’s as
well as their degree of induction upon re-exposure to the same drug dose with
some genes showing sensitized responses and others desensitized responses
(Alibhai et al., 2007; Green et al., 2008; Hope et al., 1994; Renthal et al., 2008).
In contrast, numerous other genes, such as those encoding CDK5 (cyclindependent kinase 5), several NFκB (nuclear factor κB) subunits, SIRT2 (sirtuin
2), PSD-95 (postsynaptic density protein of 95 kD), and BDNF (brain-derived
neurotrophic factor), are consistently induced only by chronic drug experience,
and some even increase further over several weeks of withdrawal after the last
drug experience (Alibhai et al., 2007; Bibb et al., 2001; Green et al., 2008; Grimm
et al., 2003; Hope et al., 1994; Renthal et al., 2008; Renthal et al., 2009; Russo
et al., 2009; Yao et al., 2004).
These complex patterns of transcriptional regulation point to the need to
identify the underlying mechanisms responsible for altering a gene’s ‘inducibility’
and those capable of stably influencing transcription for prolonged periods. In
focusing on psychostimulant-induced changes in the NAc, recent evidence has
suggested that epigenetics—a molecular translator that interprets diverse
environmental stimuli into changes in gene expression via the regulation of
chromatin structure—contributes to drug-induced transcriptional and behavioral
changes (Kumar et al., 2005; Levine et al., 2005; Maze et al., 2010; Renthal et
al., 2007; Wang et al., 2010). I propose an integrative approach in discussing
current progress being made toward understanding how epigenetic mechanisms
4
are regulated by cocaine and other psychostimulant drugs of abuse in the NAc to
influence specific gene expression programs and how such mechanisms might
contribute to addiction-related behaviors.
Epigenetics overview
Historically, the word ‘epigenetic’ refers to a heritable phenotype not
coded by DNA itself, but by a cellular process ‘above the genome’. More
recently, epigenetics is used to refer to the extremely complex processes of
organizing the genome in a manner that allows for regulated gene expression in
the appropriate cell type upon appropriate cellular stimuli. On a molecular level,
the fundamental unit that accomplishes this feat is chromatin, which is composed
of DNA wrapped around histone octomers made up of two copies each of H2A,
H2B, H3, and H4. In the past decade, it has been appreciated that the structure
of chromatin is highly regulated by post-translational modifications (PTMs) that
occur on histones and DNA itself. Importantly, these modifications—via
regulation of chromatin structure—profoundly influence gene expression in
different ways and, since multiple PTMs can occur on a given histone octomer, it
is hypothesized that the combination of these modifications summate to influence
gene expression. Also known as the histone code hypothesis (Borrelli et al.,
2008; Kouzarides, 2007; Lee and Mahadevan, 2009; Strahl and Allis, 2000).
Highlighted in Table 1-1, are just a few well characterized examples of such
PTMs, their associated effect on gene transcription, as well as the enzymes that
5
‘write’ and ‘erase’ such modifications (reviewed in greater detail in: Renthal and
Nestler, 2008; Strahl and Allis, 2000).
DNA methylation overview
DNA methylation, or the covalent addition of methyl groups to C5-position
of cytosine residues within CpG dinucleotides, forms the basis of chromatin
structure and is a central component to gene regulation in all cells (ref bird).
DNA methylation can impact gene transcription primarily through two
mechanisms.
First,
methylation
can
physically
impede
the
binding
of
transcriptional machinery to their target sequences. One well known example is
how methylation of the CRE target sequence blocks the transcription factor
CREB from binding to promoter elements (Chen et al., 2003; Martinowich et al.,
2003). The second and probably more important way that methylation influences
transcription is via recruitment of Methyl binding domain proteins (MBDs). This
family of proteins is expressed at near histone octomer levels in neurons and
specifically bind methylated DNA to subsequently recruit other pro-repressive
histone modifying enzymes such as histone deacetylases (HDACs) (Klose and
Bird, 2006; Skene et al., 2010). In addition to regulating gene expression, DNA
methylation is required for gene imprinting, x-inactivation, suppression of
retrotransposible elements, suppression of viral gene expression, and general
chromosomal stability (Klose and Bird, 2006; Skene et al., 2010).
On a genome wide level, DNA methylation displays a curious distribution
pattern which is dependent of the frequency of CpG dinucleotides and via
6
unknown mechanisms. Statistically, CpG sequences occur throughout the
genome at a much lower frequency than expected by chance.
And overall
approximately 70% of CpGs in the genome are methylated. This low frequency
in the CpG sequence is attributed to the high susceptibility of CpGs in
undergoing spontaneous mutation (via deamination). However, there are certain
hotspots in the genome called “CpG islands” that have very high frequencies of
the CpG sequence. CpG islands are highly conserved in mammals and are
mostly located within gene promoter regions (Gardiner-Garden and Frommer,
1987; Takai and Jones, 2002). Of all mammalian genes, approximately 40%
have CpG islands and, surprisingly, most CpG islands have low levels of
methylation (Klose and Bird, 2006; Takai and Jones, 2002). It is thought that the
purpose of low promoter methylation levels is to render a gene subject to
regulation via DNA methylation. In summary, normal cells have a mosaic pattern
of DNA methylation. The genome has highly methylated individual CpG sites
along with unmethylated CpG islands.
DNA methylation has been most extensively studied in the cancer field.
Here it has been found that the mosaic pattern of DNA methlyation is
differentially affected in cancer cells. There is a genome wide hypomethylation
contrasted by hypermethylation at CpG islands. In a seminal study by Weber, a
technique called mDIP-Chip (methylated DNA ImmunoPrecipitation on Chip) was
pioneered in order to ultimately identify ~200 novel hypermethylated CpG islands
(Weber et al., 2007). Moreover, thanks to major advances in microarray
technology, recent studies have found that a much greater degree of differential
7
DNA methylation (10 fold more) surprisingly occurs in a region ~2 kb upstream
from CpG islands. These regions have been called “CpG shores” (Irizarry et al.,
2009).
The three known enzymes that methylate DNA are Dnmt1 (DNA
methyltransferase 1), Dnmt3a, and Dnmt3b. Dnmt’s mediate the ezymatic
transfer of methyl groups from S-adensyl-methionine to cytosine through an
unusual mechanism whereby a cytosine base is flipped out of helical DNA,
modified, and then replaced into the helix (Jeltsch, 2002). Aside from this
mechanism, very is little is known about the upstream mechanisms that regulate
Dnmt activity and genomic specificity. Functionally, Dnmt1 is often referred to as
the “maintenance” methyltransferase because it appears to play a role in
faithfully maintaining DNA methylation patterns in newly synthesized DNA. The
evidence that Dnmt1 primarily allows for DNA methylation to be inherited after
cell division comes from observations that Dnmt1 localizes with replication foci
during S-phase and the catalytic efficiency of Dnmt1 is 3-10 fold higher on
hemimethylated substrates compared to unmethylated DNA substrates (Bacolla
et al., 1999). On the other hand, Dnmt3a and Dnmt3b are referred to as “de
novo” methyltransferases because they establish methylation patterns at specific
sites within the genome (Okano et al., 1999). Whether these ascribed functions
(maintenance and de novo methylation) are generally applicable to all
mammalian cells is a matter of debate. In all likelihood, there is functional
crosstalk between Dnmt1 and the de novo methyltransferases. For example,
Dnmt1 and Dnmt3a are the predominant isoforms expressed in adult, non-
8
dividing neurons. On a practical level, the fact that it is expressed in non-dividing
cells suggests that Dnmt1 must have some de novo methylation activity as well.
Consistent with this notion, a recent study found that conditional knock-out of
Dnmt3a was not sufficient to reduce total brain DNA methylation levels. However,
double conditional knockout of both Dnmt1 and Dnmt3a reduces DNA
methylation levels in adult neurons (Feng et al., 2010).
The fact that double knock out of Dnmt1 and Dnmt3a reduces total levels
of DNA methylation is also of interest because it suggests that mechanisms must
exist to remove methylated DNA from the genome. DNA methylation is widely
considered to be the most persistent epigenetic modification in nature. This is
attributed to the theoretical difficulty in enzymatically removing methyl-cytosine’s
carbon-carbon bond and is also attributed to the well known role that DNA
methylation plays in permanently silencing the X chromosome in females. In
light of this, considerable debate exists as to whether an actual DNA
demethylase exists. Several publications have sprung up over the years
implicating a host of different proteins in DNA demethylation—including MBDs
and even certain Dnmt’s.
At present, the leading candidate mechanism for
demethylation involves complete removal of methylated cytosine from DNA
structure via recruitment of DNA base excision repair enzymes, called GADD45’s
(Growth arrest and DNA damage repair proteins, Ma et al., 2009; Ooi and Bestor,
2008) (Table 1-1).
9
Important caveats
There are several important caveats to consider when studying
epigenetics in brain. First, a close look at Table 1-1 illustrates that many
enzymes each have multiple histone substrates and, in fact, although not listed in
the table, it is highly likely that many have non-histone substrates as well. For
example, in addition to deacetylating H4K16 (histone H4 on Lys16), SIRT2 is well
known to also deacetylate tubulin as well as several major transcription factors
(Renthal and Nestler, 2009). Therefore, in order to understand the cellular and
behavioral role of a given enzyme and histone modification, a multifaceted
approach is absolutely crucial. A second major caveat is that most of the
discovered PTMs and their ascribed influence on transcription have been derived
from in vitro work in non-neuronal cells. Therefore, it is possible that the
presumed functions of known PTMs in influencing transcription in cultured cells
may not necessarily be the same in brain. Moreover, it is likely that unique PTMs
exist in brain. In fact, a recent proteomic study on whole brain tissue identified
196 novel histone modifications (Tweedie-Cullen et al., 2009), and a study
analyzing brain DNA identified hydroxy-methylation as a novel brain-specific
DNA modification (Kriaucionis and Heintz, 2009; Tweedie-Cullen et al., 2009). A
final caveat is that histone PTMs, although the most widely studied, are just one
of many epigenetic mechanisms. A few examples that remain virtually
unexplored in brain include histone tail clipping, histone substitution, and histone
10
sliding/remodeling, which are reviewed elsewhere (Borrelli et al., 2008; Tsankova
et al., 2007).
Epigenetic mechanisms in addiction
One of the most striking findings to date is that, despite the abundance of
histone PTMs throughout the genome, drugs of abuse as well as many other
types of environmental stimuli are capable of inducing changes in total levels of
various histone modifications, for example, as detected by western blotting or
immunohistochemistry, in specific brain regions. With respect to cocaine, most
global changes observed are consistent with a state of increased gene activation
(i.e., a more permissive state) within the NAc. Following a single cocaine
injection, total levels of acetylated histone H4 (acH4), phosphoacetylated histone
H3 (pacH3), but not acH3 alone, are increased in this brain region within 30
minutes (Kumar et al., 2005). Moreover, in a cocaine self-administration model,
both acH4 and acH3 have been found to be increased in the NAc shell, not core,
following chronic but not acute self-administration. Interestingly, in these animals,
levels of acH3, and not acH4, were found to be positively correlated with
motivation for cocaine (Wang et al., 2010). Also, after repeated cocaine
injections, a recent study found a global reduction in dimethylation of H3 at Lys9
(H3K9me2), a repressive histone modification, in the NAc (Maze et al., 2010).
In concert with these global epigenetic alterations, cocaine regulates the
expression of several chromatin modifying enzymes in the NAc in directions
generally consistent with the global modifications (see Table 1-1). For example,
11
following chronic but not acute cocaine, HDAC5 is shuttled out of the nucleus of
NAc neurons and HDAC activity is significantly reduced (Renthal et al., 2007;
Romieu et al., 2008). At the same time, phosphorylation of histone H3 at Ser10
(H3pS10) appears to be mediated via direct phosphorylation by MSK1 (mitogenand stress-activated kinase 1) as well as by nuclear shuttling of DARPP-32, a
striatal-enriched protein which indirectly regulates phosphorylation via potent
inhibition of PP1 (Brami-Cherrier et al., 2005; Stipanovich et al., 2008). Likewise,
the downregulation of H3K9me2 appears to be mediated via transcriptional
repression of G9a (also known as KMT1c), a histone methyltransferase that
catalyzes H3K9 dimethylation (Maze et al., 2010). Thus, chronic cocaine appears
to globally induce a more permissive transcriptional state in the NAc (increased
acH3, acH4, pacH3; decreased H3K9me2), perhaps via differentially affecting
histone modifying enzymes that activate (MSK1, DARPP-32, CBP) and repress
(HDAC5, G9a) transcription.
Despite the fact that several chromatin modifying enzymes are regulated
in the NAc by cocaine, it is still surprising that the absolute levels of PTMs are
altered because modifications such as histone acetylation and methylation are
known to occur throughout the genome. Thus, in order to have an absolute
change, sweeping alterations must occur throughout vast genomic loci despite
the fact that many DNA microarray studies of NAc from cocaine-treated animals
have found that only a relatively small number (~100’s) of specific genes are
differentially expressed. Moreover, many specific gene promoters display
changes in histone acetylation and methylation after cocaine that go in the
12
direction opposite of the global modifications (Renthal et al., 2009). Thus, further
work is needed to understand the physiological significance of these global
changes in epigenetic PTMs.
Insights from genome-wide regulation of gene expression and chromatin
structure
Our laboratory recently utilized ChIP-chip (chromatin immunoprecipitation
followed by promoter chip) technology to investigate the genome-wide binding
profile of chronic cocaine-induced changes in the mouse NAc at two general
modifications associated with gene activation and one modification associated
with gene repression. The antibodies used in this study referred to as ‘acH3’,
‘acH4’, and ‘meH3’ each recognize multiple PTMs: polyacetylated histone H3
(K9, K14), polyacetylated H4 (K5, K8, K12, K16), and dimethylated H3 at two
sites (K9, K27), respectively (Renthal et al., 2009).
This study reported several key findings for epigenetic regulation in brain.
An obvious yet often overlooked concept is the importance and complexity of
location in analyzing the epigenetic landscape of a gene’s promoter. Here,
analysis of the averaged genome-wide spatial binding profile revealed a distinct
binding distribution pattern for each histone modification. In general, enrichment
of acH3 and acH4 occurs within 500 base pairs (bp’s) of a gene’s transcription
start site (TSS), with acH4 having a particularly sharp peak at the TSS (Renthal
et al., 2009). Conversely, NAc meH3 enrichment has a broad bimodal distribution
with maximal peaks at -1500 bp and +400 bp away from the TSS (Renthal et al.,
13
2009; Wilkinson et al., 2009). Such overt differences in spatial binding profiles
have important implications. For example, a ChIP experiment that concludes
robust acH3/H4 regulation and the lack of differential binding of meH3 may turn
out to be a false negative simply based on the genomic region in which the IP
(immunoprecipitation) and PCR reaction are focused. Moreover, based on
individual gene promoter plot analyses, several examples exist, such as the
CART promoter, in which, depending on the physical location analyzed, it is
possible to observe an increase, decrease, or no change in meH3 binding (Fig 11B). This highlights the importance of genome wide studies of chromatin
modifications and the need to extensively analyze different locations along the
landscape of a given gene promoter.
Another major finding of the Renthal et al., 2009 study was the presence
of extensive chromatin modifications found throughout the genome. With rigorous
statistical criteria, roughly 1000 gene promoters for acH3, 700 for acH4, and 900
for meH3 were found to have increased promoter binding under cocaine-treated
conditions relative to controls (Fig. 1-1A). These values are consistent with the
aforementioned observation that absolute levels of histone PTMs are altered by
cocaine. However, these findings raise a paradox: many more gene promoters,
almost one order of magnitude more, appear to be cocaine-regulated at the
chromatin level compared to the typical number of differentially expressed mRNA
transcripts that have been identified under similar cocaine treatment paradigms
(Maze et al., 2010; McClung and Nestler, 2003; Renthal et al., 2007; Yao et al.,
2004). One explanation for this disparity may lie in differences in the sensitivity of
14
the two techniques. Alternatively, the much higher degree of chromatin regulation
may suggest that each histone modification may not directly regulate steadystate mRNA levels, but rather indirectly influences transcription to subsequent
stimuli.
An important related discovery is that there is relatively little overlap
among the three different histone marks studied at the same gene promoters
(Renthal et al., 2009). This is somewhat surprising, since coincident regulation
might have been expected: for example, increased acH3, increased acH4, and
decreased meH3 at a particular activated gene. However, in this study only about
20% of genes regulated by acH3 were commonly regulated by acH4. Even more
striking, only ~1% of hypermethylated ‘repressed’ genes (9 out of 889) show
hypoacetylation (Fig 1-1A). Taken together, these findings suggest that acH4,
acH3, and meH3 each have distinct functions that regulate mostly unique
transcriptional programs.
In support of these findings, a recent genome wide microarray study,
comparing the ability of a challenge dose of cocaine to regulate gene expression
in the NAc of cocaine-naïve animals to those treated previously with chronic
cocaine, provides large scale evidence for at least two distinct patterns of gene
regulation—“desensitized” and “primed” gene expression (Maze et al., 2010). A
subset of genes, referred to as desensitized, are induced by acute cocaine and
exhibit attenuated induction after chronic cocaine. This blunted induction persists
for at least one week, as observed by the lack of effect of a challenge injection in
chronic cocaine withdrawn animals (Fig 1-1C). In contrast, the large majority of
15
genes that are induced by chronic cocaine are not induced by the initial cocaine
exposure, represent primed genes. Indeed, approximately 2-3 fold more genes
are significantly upregulated in the NAc by chronic cocaine (~275 genes) as well
as in cocaine withdrawn animals (~210 genes) compared to those upregulated by
initial cocaine exposure (~90 genes) (Maze et al., 2010). From an epigenetic
point of view, these findings suggest that throughout the course of acute to
chronic cocaine injections, many genomic loci accumulate specific epigenetic
‘hits’ that render them poised for greater induction. Further, the fact that this
primed state of gene inducibility remains present after a week of withdrawal from
cocaine suggests a prolonged underlying epigenetic influence on transcription. In
summary, this microarray study identified distinct patterns of transcriptional
regulation which are consistent with our ChIP-chip data, which revealed
extensive, mostly non-overlapping patterns of cocaine-induced promoter binding
for acH3, acH4, and meH3.
Key concepts in psychostimulant regulation of chromatin structure
Indeed, analysis of the chromatin state and mRNA expression of many
genes reveals evidence for distinct epigenetic influence in mediating both gene
desensitization and gene priming (Fig. 1-2).
Epigenetic basis of gene priming. There are several well established
examples of genes that are induced in the NAc by chronic, but not acute, cocaine
administration (Maze et al., 2010; McClung and Nestler, 2003). Specific
16
examples for which the chromatin structure has also been extensively studied
include the genes encoding BDNF, CDK5, NFκB (p65 subunit), and SIRT2 (Fig
1-2A). ChIP studies have found that genes which are primed to be induced
develop increased acH3 and no change in acH4 in response to chronic and not
acute cocaine injections (Kumar et al., 2005; Renthal et al., 2009; Russo et al.,
2009; Wang et al., 2010). Interestingly, levels of H3K9me2 and its associated
enzyme, G9a, exhibit increased binding at 1 hour after cocaine administration,
suggesting a possible role in blockade of acute induction of primed genes (Maze
et al., 2010). However, with chronic injections, both H3K9me2 and G9a binding
are reduced, consistent with the notion that this loss of methylation on H3K9 may
facilitate acetylation on H3K9 and other associated lysine residues and promote
gene inducibility (i.e., priming). Priming is also associated with increased
recruitment of SWI-SNF chromatin remodeling factors (e.g., BRG1) to the primed
gene promoters (Kumar et al., 2005).
Less is known about whether steady state mRNA levels of genes primed
for induction remain upregulated or return to baseline in between cocaine
exposures, and about what happens to the chromatin state of these genes over
this time frame. For two genes known to have prolonged regulation, CDK5 and
BDNF, increased acH3 binding was found to persist for at least one week
following the last drug injection (Kumar et al., 2005). Presumably, H3K9me2
remains reduced although this remains to be proven. In all likelihood, different
subclasses of primed genes exist—those with a prolonged increase in steadystate levels, such as BDNF, and those whose mRNAs return to baseline levels
17
but remain primed for greater induction (sensitization) during re-exposure to
drug, as illustrated in Fig. 1-1C.
Epigenetic basis of gene desensitization. The most well characterized
example of gene desensitization is the gene encoding c-Fos, which exhibits
desensitized induction in response to repeated cocaine or amphetamine
administration, and shows suppressed basal expression levels over several days
of withdrawal as well as enhanced desensitization to a challenge dose of the
drug (Renthal et al., 2008). Unlike known primed genes, acH4 and not acH3
appears to play a key role in gene desensitization. At the c-Fos promoter, acH4
is dramatically induced by acute but not chronic drug exposure (Kumar et al.,
2005). Another interesting observation lies in the temporal dynamics that occur in
response to initial drug exposure, when pacH3 precedes both peak mRNA and
acH4 levels, an effect that was no longer observed with chronic drug
administration (Kumar et al., 2005). This suggests the possible influence of
pacH3 on acH4 and subsequent gene activation. Importantly, key chromatin
modifying enzymes found to be associated with the c-Fos promoter following
chronic drug administration were HDAC1 and G9a, fitting with reduced
acetylation (via enhanced deacetylation) and increased H3K9me2 and with gene
repression (Maze et al., 2010; Renthal et al., 2008). A major goal of future
research is to determine whether other genes that display desensitization exhibit
similar chromatin mechanisms as observed for c-Fos.
18
Interplay between transcription factors and epigenetic mechanisms.
Another crucial aspect of chromatin dynamics to consider is how transcription
factors and the overall transcriptional machinery interact with epigenetic
modifications to ultimately influence transcription. In the addiction field, one of the
most well studied examples of such interactions is the transcription factor, ΔFosB
(Nestler et al., 1999; Nestler, 2008). Genome wide overexpression studies have
suggested that ΔFosB plays a dual role in repressing and activating transcription
of cocaine-regulated genes (McClung and Nestler, 2003; Winstanley et al.,
2007). As shown in Fig. 1-2, ChIP studies of NAc from chronically treated
animals find that ΔFosB is indeed bound to both primed and desensitized gene
promoters and, in each case, ΔFosB is associated with a completely different
entourage of modifications and chromatin modifying enzymes. For example, with
primed genes, ΔFosB recruits the transcriptional activator, BRG1, whereas with
desensitized genes HDAC1 is recruited to the c-Fos gene via ΔFosB (Kumar et
al., 2005). Adding to this complexity, it appears that G9a and ΔFosB interact in a
complex autoregulatory feedback loop. After acute cocaine, G9a is bound to, and
hypermethylates H3, at the FosB promoter. This hypermethylation functionally
attenuates cocaine induction of FosB gene expression, as was observed with
G9a overexpression. However, due to ΔFosB’s extraordinarily long half life,
ΔFosB accumulates in the cell and then represses mRNA levels of G9a and
reduces total levels of H3K9me2, both of which ultimately appear to reduce G9a
and H3K9me2 binding at the FosB promoter during cocaine withdrawal—thereby
allowing ΔFosB to potentiate its own induction (Maze et al., 2010).
19
Much more work is needed to understand this complex regulation,
particularly within the context of the complex behavioral parameters that define
an addicted state. For example, it will be critical in future investigations to
delineate the role played by these numerous desensitized and primed genes (in
the NAc and elsewhere) in different phases of addiction, for example, the
decision to take drug initially, the transition from casual drug use to addiction,
and the craving and risk for relapse that occur during withdrawal periods.
Role of epigenetic mechanisms in drug-related behaviors
As stated previously, biochemical and genome-wide evidence suggests
that chronic cocaine administration causes chromatin structure to be in a
generally more permissive state. This is evidenced by a reduction in total levels
of the repressive histone mark, H3K9me2, as well as an increase in total levels of
acH3 and acH4. On a genome-wide scale, in addition to each of the
aforementioned modifications being heavily regulated by chronic cocaine at
literally thousands of gene promoters, 2-3 fold more genes are activated by
chronic cocaine compared to acute cocaine. Behaviorally, increasing evidence
suggests that the permissive chromatin structure induced by chronic cocaine is a
pathological adaptation to the rewarding effects of the drug (Table 1-2). For
example, mimicking this ‘permissive state’ by systemic administration of
trichostatin A (TSA) or intra-NAc delivery of suberoylanilide hydroxamic acid
(SAHA), which are both selective class I and class II HDAC inhibitors, increase
cocaine conditioned place preference, which provides an indirect measure of
20
cocaine reward (Kumar et al., 2005; Renthal et al., 2007). In addition, intra-NAc
pharmacological blockade as well as intra-NAc conditional knock-out of another
repressive modification, G9a (H3K9me2), also increases cocaine place
conditioning (Maze et al., 2010). Conversely, experimental manipulations that
promote gene repression, such as intra-NAc overexpression of HDAC4, HDAC5,
or G9a, reduce cocaine place conditioning. These effects appear to depend
specifically on enzymatic activity of these enzymes, since catalytically dead
mutants of HDAC5 or G9a (G9a-H1093K) have no effect on cocaine reward, and
since overexpression of wild type HDAC5 co-administered with TSA completely
blocks HDAC5’s effect on behavioral responses to cocaine. Furthermore, these
effects on cocaine reward are specific to certain enzymes and are not
generalizable, since, for example, NAc-specific overexpression of HDAC9—an
enzyme that is not cocaine regulated, but is expressed in the NAc—does not
have any effect on cocaine reward (Renthal et al., 2007).
It is important to note that this general notion that promoting a more
permissive chromatin state does not always produce increased cocaine reward.
For example, resvertitrol, a sirtuin (class III HDAC) agonist, and sirtintol, a sirtuin
antagonist, unexpectedly increase and decrease cocaine reward, respectively
(Renthal et al., 2009). This is surprising since SIRT1, a key target of these
agents, is a major component of transcriptional repression complexes (Finkel et
al., 2009). Also, despite both manipulations reducing H3S10 phosphorylation,
MSK1 knockout increases cocaine reward whereas DARPP-32 S97A knock-in
decreases cocaine reward (Brami-Cherrier et al., 2005; Stipanovich et al., 2008).
21
However, while sirtuins, MSK1, and nuclear DARPP-32 have effects on histones,
they also have a multitude of non-histone substrates, which makes it difficult to
establish a solid link between their effects on chromatin and their resultant
behavioral actions—an important concept.
From a theoretical standpoint, since chromatin modifications such as
histone acetylation are affected differently by acute compared to chronic cocaine,
one would expect that experiments that manipulate chromatin dynamics at
different times of drug administration (i.e., before receiving initial cocaine, after
receiving chronic cocaine, or during cocaine extinction) may have different
results. A limited number of experiments have specifically addressed this
question and support this concept (Table 1-2). All of the aforementioned
experiments have analyzed baseline manipulations that occur during behavioral
training and testing. However, in one experiment, it was found that loss of
HDAC5 and not HDAC9 results in lasting sensitization to cocaine reward:
HDAC5 knock-out had no effect on cocaine place conditioning at baseline but
sensitized the ability of prior cocaine exposure to enhance place conditioning
(Renthal et al., 2007). Furthermore, a series of key cocaine self-administration
studies have found that the timing of HDAC inhibition is likely to be crucial. For
example, Romieu et al (2008) found that the daily systemic (IV) administration of
TSA during the initial acquisition phase of cocaine self-administration reduced
instrumental responding including reduced break point in a progressive ratio test,
whereas another study found that a single systemic injection of sodium butyrate
(a broadly acting and non-specific HDAC inhibitor among many other actions)
22
during the maintenance phase significantly enhanced administration behavior
(Sun et al., 2008). Similarly, a recent study found that TSA and SAHA infusions
in the NAc shell, but not core, greatly enhanced motivation for cocaine during the
chronic phase of cocaine self-administration (long-term reinforcement of cocaine
self-administration for more than 30 days) (Wang et al., 2010). Finally, Malvaez
and colleagues found that systemic sodium butyrate injections given after CPP
extinction and reinstatement training resulted in an enhancement of extinction
and reduced reinstatement (Malvaez et al., 2010). Based on this range of
interesting results, it is clear that, similar to ChIP and mRNA studies, acute
exposure to cocaine imposes different effects compared to chronic cocaine, and
presumably the environmental context also imposes unique effects—all of which
are simultaneously affected by these manipulations.
Depression and epigenetic mechanisms (Brief overview)
Depression is a major cause of worldwide morbidity. It is estimated that
the lifetime prevalence in the United States of developing major depression is as
high as 17%. Vulnerability to depression has a strong genetic component
estimated at 40-50% (Krishnan and Nestler, 2008). However, even after years of
research and major advances in genome sequencing, the genetics of depression
remains poorly understood. Conversely, it is clear that environmental factors
such as chronic stress, emotional trauma, and several medical illnesses are
major contributors to depression. Given the prevalence it is not surprising that
antidepressants are consistently among the top prescribed medications in
23
America. What remains surprising is the paucity in understanding of how
medications like fluoxetine (a commonly perscribed SSRI) exert antidepressant
responses only after prolonged (weeks of) administration. Similar to addiction,
the pathophysiology of depression clearly involves several brain structures.
Because of its central role in influencing mood and reward, one structure of
particular focus in depression is the nucleus accumbens (NAc). Also similar to
drug addiction, a leading hypothesis for the basis of depression suggests that
persistent changes in gene expression ultimately change neural function and
behavior (Covington et al., 2009).
Such prolonged alterations in NAc gene expression are well documented
in both human depression and rodent models of depression (Krishnan et al.,
2007; Wallace et al., 2009). Compared to addiction research, much less is known
about the basis for these changes in gene expression, however increasing
evidence similarly points to epigenetic mechanisms. For example, in a ChIPChip study looking at the role of repressive histone methylation in two different
models of depression, my colleagues and I found that gene promoters are
extensively and similarly methylated in these models. Using heatmap analysis, I
also found that both chronic antidepressants and resiliency to stress completely
block or reverse stress-induced methylation patterns (Wilkinson et al., 2009).
Ultimately, this study suggests that epigenetic mediated gene repression is a
major factor in promoting depressive behavior and that mechanisms that promote
gene activation should have therapeutic efficacy in treating depression.
Consistent with this notion, a study by Covington found that continuous intra-NAc
24
infusion of HDAC inhibitors (MS-275 or SAHA, both of which promote gene
activation) function as antidepressants. Next, in a microarray study comparing
the expression profile of MS-275 to the standard antidepressant, fluoxetine, I
found that both fluoxetine and HDAC inhibition reverse stress induced gene
expression through a combination of similar and novel transcriptional
mechanisms (Covington et al., 2009). One important next step will be to further
identify whether other repressive modifications, such as DNA methylation, play a
similar role in the NAc in promoting depressive behavior.
DNA methylation in neuroscience
In recent years, the role of DNA methylation in the CNS has gained
considerable attention. As previously mentioned, this interest is largely due to the
observation that the DNA methyltransferases, Dnmt1 and Dnmt3a are
unexpectedly highly expressed in adult, post-mitotic neurons (Feng et al., 2005).
Moreover, in the past decade, DNA methylation has been heavily implicated in
several
neurological
disorders,
particularly
psychiatric
diseases
with
developmental etiology. The most convincing data come from several published
findings which have shown that various mutations in the methyl-binding domain
protein, MeCP2, cause Rett syndrome (an autism spectrum disorder) (Zoghbi,
2009). These mutations do not result in neuronal cell death and, amazingly,
genetic rescue with Wt MeCP2 has been shown to reverse Rett associated
neurological defecits (Guy et al., 2007). Two other mental retardation disorders,
Fragile X and ICF syndromes, also arise from abnormal DNA methylation. Fragile
25
X mental retardation is associated with expansion of a single trinucleotide gene
sequence (CGG) on the X chromosome. The severity of the disorder is linked to
the extent of CGG repeats and the degree of DNA methylation at these repeats
which
ultimately
silence
the
expression
of
the
FMR1
gene.
ICF
(Immunodeficiencey, Centromeric region instability, Facial anomalies) syndrome
is a rare autosomal recessive disorder caused by mutations in the DNMT3b gene
which result in genome wide hypomethylation. Finally, a series of association
studies have led to the hypothesis that schizophrenia may be caused by aberrant
DNA methylation. For example, high doses of the amino acid l-methionine (and
Dnmt coenzyme) were found to exacerbate schizophrenic symptoms (Cohen et
al., 1974; Grayson et al., 2009). Moreover, levels of SAM (s-adenosylmethionine)
and Dnmt1 have been found to be elevated in the frontal cortex from
schizophrenic post-mortem brain tissue (Guidotti et al., 2007; Veldic et al., 2005).
Third, elevated levels of Dnmt1 correlate with increased methylation and reduced
mRNA expression of GAD67 and reelin in schizophrenic tissue (Grayson et al.,
2005). These studies have also been extended to mouse models. Interestingly,
as originally associated in humans, it was demonstrated that chronic systemic Lmethionine administration causes hypermethylation of GAD67 and reelin in
frontal cortex in mice (Dong et al., 2005). In summary, aberrant DNA methylation
is implicated in autism, mental retardation, and schizophrenia. Of note, these are
all neurodevelopmental disorders thought to be due in part to improper neuronal
plasticity.
26
Several studies on the role of DNA methylation in hippocampus also
support the notion that methylation regulates neuronal plasticity, particularly
synaptic plasticity. In hippocampal culture, Dnmt inhibition causes a marked
reduction in number of functional synapses, as measured by a reduction in
miniature excitatory post-synaptic current (mEPSC) frequency (Nelson et al.,
2008). Other studies in hippocampal slices have found that DNMT inhibition
blocks long term potentiation (LTP) (Levenson et al., 2006). More recently, it has
been shown that double (and not single) conditional knock-out of Dnmt1 and
Dnmt3a similarly blocks LTP (Feng et al., 2010). Behaviorally, Dnmt3a mRNA
was found to be transiently upregulated immediately following fear conditioning.
And both Dnmt inhibition and double cKO of Dnmt1/Dnmt3a impairs memory
formation in several tested learning and memory tasks (Feng et al., 2010; Lubin
et al., 2008; Miller and Sweatt, 2007).
No studies so far have explored the role of DNA methylation in the
pathophysiology of addiction or depression. By focusing on the NAc, my thesis
provides the first look at the role of DNA methylation in these devastating
psychiatric
diseases.
In
chapter
2,
I
first
explore
whether
DNA
methyltransferases are regulated by chronic cocaine or by chronic social defeat
stress. I then developed and used complementary pharmacological and genetic
techniques to manipulate DNA methylation in order to test how this regulation
affects reward behavior and synaptic plasticity. Chapter 3 describes an extensive
study, still underway, aimed at identifying the genes regulated by DNA
methylation in the NAc. And the final chapter concludes my thesis with a detailed
27
discussion of how my findings relate to other studies on addiction and
depression, the limitations of these findings, as well as future studies that will
further unravel the role of epigenetics in addictive and depressive disorders.
28
CHAPTER 1 TABLES
Table 1-1. Histone modifications, their function, and their respective
enzymes.
Modification
Phosphorylation
Effect on gene
expression
↑
H3pS10
"Writers" (enzymatic
addition)
kinases
AURKB, MSK1
Ubiquitination
↑
Ub ligases (RING2)
Sumolyation
↓
SUMO E2s/E3s? (UBC9)
Acetylation
H3K9ac
↑
↑
me1↑ , me2/3 ↓
↓
↑
↑
KATs
2a (GCN5), 2b, 12
2a-b, 3a (CBP), 3b (p300),
6a, 6b
5, 8
KMTs
2a-h, 7
1a-f (1c=G9a/EHMT2), 8
6
3a-c
4
?
↓
PRMTs (1-8)
DNMT1, DNMT3a, 3b
H3K14ac
H4K16ac
Lysine Methylation
H3K4me3
H3K9me1/2/3
H3K27me3
H3K36me3
H3K79me3
Arginine
Methylation
DNA methlyation
↑↓
"Erasers"
(enzymatic removal)
Phosphatases
PP1, DARPP32
(indirectly via PP1
regulation)
Ub protease
(USP16)
SUMO protease
(SUSP17)
HDACs (1-11),
HDAC4,5, 9
"
"
Sirt2
KDMs
1, 2a, 5a-d
1, 3a-b, 4a-d
6a-b
2a-b, 4a-c
?
JMJD6
Gadd45a,b,g ?
List of histone modifications, their known effects on transcription and examples of enzymes that
catalyze their "writing" (enzymatic addition) or "erasing" (enzymatic removal)
↑ = increased transcription, ↓ = decreased transcription; Blue = examples of enzymes in which
cocaine-induced biochemal regulation has been identified and it's behavioral significance has
been accessed
29
Table 1-2. Role of Epigenetic modifications in cocaine reward behaviors
Experimental
manipulation
Histone
acetylation
Sodium Butyrate HDACi
Sodium Butyrate - "
Sodium Butyrate - "
TSA - Class I & II
HDACi
TSA - "
TSA - "
SAHA - Class I & II
HDACi
SAHA - "
resveritrol - Class
III (sirtuin) HDAC
ago
Route of
administration
IP, preceeding
drug injection
IP, after drug
training
IP, preceeding
drug injection
IP, preceeding
drug injection
IV, preceeding
drug injection
Conditioned
place
preference
Locomoto
r
sensitizati
on
↑
extinction
↑
↑
↓
Intra-NAc shell
Intra-NAc shell
Intra-NAc cont.
delivery
Self-admin.
↓ acquisition
phase of SA
↑ motivation
for cocaine
↑ motivation
for cocaine
↑
IP, preceeding
drug injection
Reference
Kumar et al.,
2005
Malvaez et
al., 2009
Sun et al.,
2008
Kumar et al.,
2005,
Romieu et
al., 2008
Wang et al.,
2010
Wang et al.,
2010
Renthal et
al., 2007
Renthal et
al., 2009
sirtinol - Class III
(sirtuin) HDACi
HDAC4
overexpression
Intra-NAc cont.
delivery
Intra-NAc,
HSV/AAV
HDAC5 KO
HDAC5 KO +
HDAC5 overexp.
knock out
knock out +
intra-NAC, HSV
HDAC9 KO
knock out
-
HDAC5 ovexp.
Intra-NAc, HSV
↓
HDAC5-cat ovexp.
HDAC5-dMef
ovexp.
HDAC5 ovexp. +
TSA
HDAC9
overexpression
Intra-NAc, HSV
-
Intra-NAc, HSV
Intra-NAc, HSV
+ IP TSA
↓
Intra-NAc, HSV
-
CBP het/KO
Histone
Phosphorylation
knock out
MSK1 KO
knock out
↓ SA, dose
response
curve
↓ motivation
for cocaine
↓
↓
↑ with
reexpsoure
"- with
reexpsoure
-
↑
Renthal et
al., 2009
Kumar et al.,
2005,
Renthal et
al., 2007
Renthal et
al., 2007
Renthal et
al., 2007
Renthal et
al., 2007
Renthal et
al., 2007
Renthal et
al., 2007
Renthal et
al., 2007
Renthal et
al., 2007
↓
Levine et al.,
2005
↓
BramiCherrier et
30
DARPP-32 S97A
Histone
methylation H3K9me2
G9a
overexpression
G9a-H1093k
overexpression
G9a inhibition (BIX)
G9a cKO
DNA methylation
DNMT inhibition
DNMT
overexpression
red= more
permissive state
green= more
repressive state
Knock -in
↓
Intra-NAc, HSV
↓
Intra-NAc, HSV
Intra-NAc cont.
delivery
Intra-NAc, AAVCRE
-
↓
↑
↑
???
???
red=behavior associated w/ increased
cocaine reward
green= behavior associated with decreased
cocaine reward
al., 2005
Stipanovich
et al., 2008
Maze et al.,
2010
Maze et al.,
2010
Maze et al.,
2010
Maze et al.,
2010
31
32
Figure 1-1: Summary of genome wide analysis of cocaine regulation of
chromatin structure and gene expression. A) Chronic cocaine causes
extensive genome-wide chromatin modifications marked by little overlap. Venn
diagrams from ChIP-chip studies with acetylated H3 (acH3), acetylated H4
(acH4), and methylated H3 (meH3) from nucleus accumbens (NAc) in mice that
received chronic cocaine (24 hours after 7 daily cocaine injections at 20 mg/kg
dose) (Renthal et al., 2009). Compared to traditional DNA microarray studies that
look for genes with altered steady state mRNA levels under similar conditions, a
much greater number of gene promoters are significantly regulated by each
analyzed histone modification and, yet, a relatively small number of gene
promoters share co-regulation of more than one modification suggesting that
each analyzed modification has distinct functions. B) Example of promoter plots
of two representative genes, Cart and Per1, from the Venn diagram illustrating
spatial complexity in chromatin regulation. Y-axis represents fold change in
binding relative to saline treated mice and the x-axis represents the physical
location of binding on the indicated gene promoter relative to its transcription
start site (TSS). The plots illustrate the complex patterns of acH3, acH4, and
meH3 on these two representative gene promoters. C) Analysis of cocaineinduced gene expression reveals two patterns of gene induction: desensitized
(black line in acute row) and primed genes (purple lines in chronic and withdrawn
columns). Scaled heatmaps (*) show the number and pattern of cocaineupregulated gene expression in NAc at 1 hour after a challenge injection of
cocaine (20 mg/kg) in three groups of animals: Acute (no prior cocaine
33
exposure), Chronic (7 daily cocaine injections examined 1 hour after the last
injection), or Withdrawn (7 daily cocaine injections followed by 1 week of
withdrawal before the challenge injection). The top series of heatmaps show the
genes upregulated in the Acute group and how those same genes are regulated
in the other two groups. Likewise, the middle and lower series of heatmaps show
the genes upregulated in the Chronic and Withdrawal groups, respectively, and
how those genes are regulated in the other two groups. Notably, a small but
significant number of acutely induced genes are no longer induced in animals
with prior chronic cocaine exposure (desensitized genes, black bar***), whereas
a larger number of genes that are induced after chronic cocaine lack induction by
acute cocaine (primed genes, purple bars***). Adapted from Maze et al., 2010.
34
35
Figure 1-2: Distinct patterns of cocaine-induced gene induction correspond
to unique patterns of chromatin regulation. Graphs in the top of A and B
display patterns of mRNA expression over time, with the underlying epigenetic
binding profile depicted in the bottom tables. Promoter binding profiles are
displayed in heatmap style (cocaine relative to saline, with red = increased
binding, black = no change, green = reduced binding). The x-axis is divided into 4
sections that highlight mRNA and chromatin changes that occur in response to
initial cocaine exposure, in response to a cocaine challenge after prior chronic
drug exposure, during drug withdrawal, and in response to a subsequent
challenge dose of cocaine. Of note, since chronic injections are given once daily,
one can infer that chromatin changes that occur during early withdrawal (24
hours) are presumed to provide insight into events that also occur prior to the last
chronic cocaine injection. A) Epigenetic basis of gene priming. Genes that are
primed to be induced eventually develop increased acH3 and reduced H3K9me2
binding during a chronic course of cocaine administration. In contrast, during
initial cocaine exposure—when there is no mRNA induction—the repressive
histone methyltransferase, G9a, is induced and binds to and mediates H3K9me2
binding to nearby promoter regions. B) Epigenetic basis of gene desensitization.
Genes that are desensitized exhibit prominent induction of acH4 binding during
initial cocaine exposure, which is lost with chronic treatment. This may be due to
increased binding of HDAC1. As well, G9a shows increased binding during acute
and withdrawal time points. This is correlated with increased H3K9me2 during
late withdrawal. Interestingly, the transcription factor, ΔFosB, is bound to both
36
types of genes during cocaine withdrawal, suggesting a dual role in mediating
gene repression and gene activation. An important caveat is that these
mechanisms of gene priming and desensitization are based on the analysis of a
relatively small number of cocaine-regulated genes and must be refined as many
more such genes are characterized. N/A, information not available.
37
CHAPTER 2
DNMT3A REGULATES EMOTIONAL BEHAVIOR AND SPINE PLASTICITY IN
THE NUCLEUS ACCUMBENS.
ABSTRACT
Despite abundant expression of DNA methyltransferases (Dnmt’s) in
brain, the regulation and behavioral role of DNA methylation remain poorly
understood. We find that Dnmt3a expression is regulated in nucleus
accumbens (NAc) by chronic cocaine and chronic social defeat stress.
Moreover, NAc specific manipulations that block DNA methylation
potentiate cocaine reward and exert antidepressant-like effects, whereas
NAc specific Dnmt3a overexpression attenuates cocaine reward and is prodepressant. On a cellular level, we show that chronic cocaine selectively
increases thin dendritic spines on NAc neurons and that DNA methylation
is both necessary and sufficient to mediate these effects. These data
establish the importance of Dnmt3a in the NAc in regulating cellular and
behavioral plasticity to emotional stimuli.
38
INTRODUCTION
Chronic cocaine and chronic social defeat stress alter gene expression,
neuronal plasticity, and ultimately behavior, and we and others have implicated
chromatin remodeling in playing a key role in regulating these events in the
nucleus accumbens (NAc), a key brain reward region (Covington et al., 2009;
Kumar et al., 2005; Levine et al., 2005; Malvaez et al., 2010; Maze et al., 2010;
Renthal et al., 2007; Renthal et al., 2009; Schroeder et al., 2008; Tsankova et al.,
2007). However, work to date has focused primarily on more labile epigenetic
modifications such as histone acetylation and methylation. Given the persistent
nature of addiction, an intriguing possibility is whether more stable epigenetic
modifications, such as DNA methylation, can more persistently influence gene
expression in the NAc to maintain this behavior.
Despite abundant neuronal expression of DNA methyltransferases
(Dnmt’s)(Feng et al., 2005), little is known about the function of DNA methylation
in brain. Behavioral studies suggest that DNA methylation is required for
hippocampal-dependent memory formation (Feng et al., 2010; Miller and Sweatt,
2007). Pharmacological inhibition of DNA methylation blocks long-term
potentiation in hippocampus (Levenson et al., 2006) and this effect depends on
the activity of both Dnmt1 and Dnmt3a (Feng et al., 2010). Moreover, Dnmt
inhibition dramatically reduces functional synapses formed by cultured
hippocampal neurons as measured by a reduction in mEPSC frequency (Nelson
39
et al., 2008). Together, these studies point to DNA methylation playing a crucial
role in hippocampus in memory formation and associated effects at the synapse.
In the present study, we focus on the NAc to test if these concepts
regarding DNA methylation extend to drug addiction and depression models. By
analyzing the role of DNA methylation in the context of both chronic cocaine (a
rewarding stimulus with persistent effects) and chronic social defeat stress (an
aversive stimulus with persistent effects), these studies complement one another
by establishing the influence of this lasting epigenetic modification in the NAc
across a spectrum of complex behaviors in which this brain region plays an
important
role.
Toward
this
goal,
we
identify
which
specific
DNA
methyltransferases (Dnmt1, Dnmt3a, or Dnmt3b) are regulated in chronic
cocaine and chronic stress paradigms, whether manipulating DNA methylation
affects addictive- and depressive-like behavior, and whether DNA methylation
affects synaptic plasticity in the NAc.
40
METHODS
Animals
We used adult male c57bl6j mice (Jackson) and Long-Evans rats (Charles
River). Animals were habituated in our facility 1 week prior to experimentation
and were housed on a 12 hr light-dark cycle with access to food and water ad
libitum. All animal experiments were approved by IACUC’s of Mount Sinai School
of Medicine and UT Southwestern.
Drugs
For chronic cocaine (Sigma) experiments, we used a standard cocaine
injection paradigm (Kumar et al., 2005; Maze et al., 2010; Renthal et al., 2007;
Renthal et al., 2009): 7 daily intraperitoneal (IP) injections of 20 mg/kg cocaine.
For acute experiments, mice were injected for 6 days with saline before a
cocaine injection on day 7. Controls were injected with an equivalent volume of
saline. For spine analysis, we used an established injection schedule known to
increase spine density, which consists of five 20 mg/kg injections. This protocol is
shortened to coincide with the duration of HSV-mediated overexpression which
wanes 6 days post-op (Barrot et al., 2002; Russo et al., 2009).
For methionine (MP Biochemicals) experiments, animals were injected
subcutaneously with 0.78 g/kg L-methionine 2x/day for 10 days. This was timed
to ensure that the minimum dose and injection duration previously shown to
increase DNA methylation in the striatum (Dong et al., 2005; Dong et al., 2008),
41
would coincide with the beginning of CPP training. During training, animals were
injected with methionine 2-3 hrs prior to behavioral experiments.
For RG108 (Sigma) experiments, we used 7-10 days of continuous (0.25
l/hr) intra-NAc delivery of 100 M RG108 dissolved in 5% hydroxypropyl βcyclodextrin vehicle (Trappsol). This dose of RG108 was found to decrease DNA
methylation in vivo and was void of evidence of neurotoxicity as assessed by
activated caspase-3 staining. RG108 was chosen over other Dnmt inhibitors,
such as 5-aza-cytidine, because 1) it inhibits methylation without incorporation
into DNA which has been shown to be linked to cytotoxicityJuttermann et al.,
1994 and 2) it is chemically stable with a mean 37°C t½ of 20 days, whereas the
37°C t½ of 5-aza-cytidine is on the order of hr (Brueckner et al., 2005). Osmotic
delivery was performed as previously described (Covington et al., 2009; Maze et
al., 2010; Renthal et al., 2007).
For fluoxetine (Tocris) experiments, we injected mice with 20 mg/kg
fluoxetine IP for 14 days, as described previously (Berton et al., 2006; Covington
et al., 2009).
Cocaine self-administration
Self-administration was performed as previously described (Noonan et al.,
2008; Spano et al., 2007). Animals had 3 hr daily access to cocaine (0.75
mg/kg/infusion) under a fixed ratio-1 (FR1) reinforcement schedule. For the 24 hr
withdrawal experiment, rats had 13 days of cocaine intake; for the 28 day
withdrawal experiment, rats had 3 weeks of intake.
42
Herpes simplex and adeno-associated virus injections
We used the bi-cistronic p1005+ HSV vector, which expressed GFP alone
or GFP with Dnmt3a. In this system, HSV infection occurs selectively in neurons.
GFP expression is driven under the human immediate early cytomegalovirus
(CMV) promoter, while the gene of interest, Dnmt3a, is driven by the IE4/5
promoter (Clark et al., 2002). We used a previously cloned mouse Dnmt3a-1
plasmid (Linhart et al., 2007). AAV-GFP or AAV-CreGFP was exactly used as
describedMaze et al., 2010. Stereotaxic surgery was performed based on
published methods (Maze et al., 2010; Renthal et al., 2007).
Conditioned place preference
We used a standard, unbiased CPP procedure (Renthal et al., 2007;
Renthal et al., 2009; Russo et al., 2009). In brief, before experimental
manipulation, animals were pretested for 20 min in a photo-beam monitored box
with free access to environmentally distinct chambers. The mice were then
arranged into control and experimental groups with equivalent pretest scores.
After experimental manipulation, mice underwent four 30 min training sessions
(alternating cocaine and saline pairing). On the test day, mice had 20 min of
unrestricted access to all chambers, and a CPP score was ultimately assigned by
subtracting time spent in the cocaine-paired chamber minus time spent in the
saline-paired chamber.
43
Cocaine-induced locomotor sensitization
We used a standard locomotor sensitization procedure (Pulipparacharuvil
et al., 2008). In brief, mice (saline habituated) were injected daily with cocaine
(IP) 30 min after being placed in standard plastic cages similar to their home
cages. Total locomotor activity was measured via photobeam breaks for 30 min
following their injection (20mg/kg).
Social defeat stress
Experimental C57bl6j mice were subjected to 10 days of social defeat
stress as previously described (Berton et al., 2006; Krishnan et al., 2007;
Krishnan et al., 2008). In summary, mice are exposed daily to an unfamiliar
aggressive CD1 mouse for 5 min. For the remaining 24 hr, the defeated mouse
was housed in a ‘protected’ compartment of the same cage where it endured
chronic stress from the CD1. On the 11th day, mice underwent a social interaction
test to verify defeat-induced social avoidance (Berton et al., 2006; Tsankova et
al., 2006). As previously published, control mice spend more time interacting with
a social “target” as compared to “no target”, whereas chronically defeated mice
spend significantly less time interacting with the target mouse compared to “no
target” (Berton et al., 2006; Krishnan et al., 2007; Tsankova et al., 2006).
For RG108 and fluoxetine experiments, we selected mice displaying
robust social avoidance—showing “Day 11” interaction times <40 sec. Then,
using baseline interaction times, we divided animals into 3 similar groups as
follows – Controls: intra-NAc vehicle (75.1 ± 3.1 sec), intra-NAc RG108 (75.4
44
±4.1 sec), or systemic fluoxetine (75.5 ± 6.1 sec); Defeats: intra-NAc vehicle
(20.5 ± 3.3 sec), intra-NAc RG108 (20.4 ±3.6 sec), fluoxetine (20.1 ± 3.1 sec).
To test for a pro-depressive phenotype, we performed a submaximal
defeat experiment as previously described (Krishnan et al., 2007; Krishnan et al.,
2008): 3 days following HSV surgery, naïve mice are subjected to 3 consecutive
5 min defeat episodes interspersed by 15 min rest periods. Mice underwent the
social interaction test the following day. In this paradigm, these stressors are not
sufficient to cause social avoidance in HSV-GFP mice as indicated by a
significant increase in “target” interaction time versus “no target” interaction time
(Krishnan et al., 2007; Krishnan et al., 2008).
Forced swim test
Immobility measures were obtained using a 2-day forced-swim test
procedure commonly used in rats (Krishnan et al., 2008; Porsolt et al., 1977).
Three days following surgery, Sprague-dawley rats were forced to swim for 15
min in plastic cylinders (20 × 45 cm). The following day, rats were tested under
identical conditions for 5 min. All sessions were videotaped and scored by a
blinded observer. Latency to immobility was defined as the time that the rat first
initiated a stationary posture that did not reflect attempts to escape.
Novel object recognition and object habituation tests
Tests were performed according to previous publications with minor
modifications (Frick and Gresack, 2003). Briefly, for the novel object recognition
45
test, mice were placed in an open field area to habituate (15 min). After 3 min of
rest, they were exposed to 3 objects for 5 min in the same arena. Each object
was placed in opposite corners of the open field arena. Mice were reexposed two
additional times to the same objects in the same locations and on the final trial,
one object was switched with a novel object. A normal mouse with intact memory
is expected to spend more time with this novel object as compared to the familiar
objects. For habituation to a novel object, we exposed mice to a single object for
150 sec and the re-exposure time was extended to one hr. Here, as mice are reexposed to this object, they are expected to become disinterested in the object,
as measured by number of object investigations.
RNA isolation, reverse transcription, quantitative PCR, and primers
Bilateral NAc punches were obtained and the tissue homogenized in
TriZol (Invitrogen). RNA was purified with RNeasy Micro columns (QIAGEN).
Reverse transcription of RNA was carried out using iScript (Biorad). qPCR was
run using approximately 5 ng of cDNA per reaction. Each reaction was run in
triplicate and quantified using the ΔΔCt method as previously described
(Tsankova et al., 2006). A complete list of primers is given below.
Global DNA methylation analysis
We used Epigentek’s Methylamp Global DNA Methylation Quantification
Ultra Kit and followed manufacturer’s instructions. This method is ideally suited
for our experiments on NAc tissue because of its ability to use small amounts of
46
input DNA and for high degree of sensitivity. Raw values were colorimetrically
quantified and total methylation levels estimated by generating a standard curve
from Epigentek’s methylated DNA standard. Values are then represented as
methylation % relative to vehicle control.
ChIP promoter analysis
Chromatin from NAc tissue punches was immunoprecipitated with an
antibody against me3K4H3 (Abcam ab1012) as described previously without
modifications (Kumar et al., 2005; Renthal et al., 2009; Wilkinson et al., 2009).
For the 24 hr experiment, samples were amplified using GenomePlex whole
genome amplification kit (Sigma), labeled with Cy3 (input) or Cy5 (me3K4H3
enriched), and hybridized to NimbleGen MM8 mouse promoter arrays with 3
biological replicates used per condition. Each replicate consisted of bilateral NAc
punches pooled from 10 mice. ChIP-chip analysis was performed as described
previously without modification (Renthal et al., 2009; Wilkinson et al., 2009).
Based on these analyses, the Dnmt3a promoter was found to have significantly
reduced binding at approximately -500 bp upstream of the transcriptional start
site. ChIP’s were then performed on additional sets of 4 and 24 hr cocaineinjected mice and PCRs were performed using primers designed around this
region as well as -2000 bp upstream.
Dendritic spine analysis (GFP immunostaining and single-cell dye filling)
47
For viral-based (GFP immunostaining) spine analysis (Fig. 2-4a), mice
were perfused with 4% PFA 4 hr following the final cocaine injection and brains
sectioned at 100 μm using a Leica vibratome. Slices were immunostained with
an anti-GFP antibody (Millipore). Sections were mounted, coded, and confocal
imaging (Zeiss LSM 710) was performed with the rater blind to experimental
conditions. We imaged secondary and tertiary dendrites of NAc medium spiny
neurons under a 100x oil-immersion objective at a resolution of 0.027 μm x-axis x
0.027 μm y-axis x 0.3 μm z. Approximately 7-10 neurons were imaged per
animal (average total dendritic length 400 μm/animal, n=5-6). Dendritic length
was measured using NIH ImageJ software and spine numbers were counted
manually by a trained observer who was also blind to experimental conditions. To
account
for
any
possible
surgical
or
sampling
bias,
we
annotated
anterior/posterior or NAc core/shell location of all images and found no significant
differences in location or region.
Because HSV-GFP (like other HSV encoded transgenes) is no longer
expressed after 7 days, we performed single-cell dye filling experiments (Fig. 24b,c). As previously described (Radley et al., 2006), random cells in the NAc
shell were impaled with a micropipette containing 5% Lucifer Yellow (Molecular
Probes) and injected with 1-10 nA of current. Slices were washed in PBS,
mounted and imaged on the confocal microscope using the same imaging
parameters as the above GFP experiment. On average, for each animal 12
dendrites from 6 neurons were imaged (average total dendritic length of 500
μm/animal, n=4-8). Spine density and spine type analysis were performed using
48
the
semi-automated
software,
NeuronStudio
(http://research.mssm.edu/cnic/tools-ns.html) (Rodriguez et al., 2008). Of note,
NeuronStudio analysis is not amenable to other dendritic images, such as GFP,
Golgi, or diolistics. NeuronStudio analyzes single-cell filled deconvolved images
in 3 dimensions (3D) and, as an extra advantage, this software uses an algorithm
of voxel clustering and Rayburst Sampling to ultimately classify spines into the 3
major morphologic types—thin, mushroom, and stubby. After NeuronStudio
processing, a human operator verifies that all spines have been appropriately
identified and manually corrects any errors in spine characterization. For the
operator, the manual aspect of analysis takes about 10 min per dendritic image.
Due to the labor intensive nature of 3D analysis, we had a separate
NeuronStudio operator for each of the 3 cell-filling experiments. For each
experiment, the NeuronStudio operator randomly analyzed coded images. Upon
finishing analysis, the spine density (as well as spine type) was calculated by
dividing the total number of spines present by the dendritic length of the
segment. Human-operated NeuronStudio was previously shown to have a range
of 68% to 90% inter-operator variability in spine density counts (Rodriguez et al.,
2008). Similarly, we found a high degree of inter-operator variability in our 3
datasets and, therefore, to control for such variability and to allow for comparison
across experiments, we normalized the spine density and spine type data from
each experiment with respect to its own control. Data are expressed as %
change in spine density relative to saline controls.
49
Statistical analysis
Statistical significance was measured using an unpaired two-tailed
Student t-test when comparing two groups. One-tailed Student t-tests were used
for pharmacological verification of changes in global methylation and 7day-4hr
spine density analysis since values are expected to fall in an expected sampling
distribution. For mRNA analysis, HSV-GFP dendritic spine analysis, submaximal
defeat susceptibility, and antidepressant tests in defeated mice, two-way
ANOVA’s were performed since experiments contained multiple groups.
Repeated measures ANOVA was used for locomotor sensitization analysis. For
ANOVAs, post-hoc comparisons were performed when appropriate.
50
RESULTS
Transcriptional regulation of Dnmt3a in NAc by cocaine
As a first step in determining the role of DNA methylation in cocaine
action, we performed quantitative PCR (qPCR) on NAc tissue of mice treated
acutely or chronically (7 injections) with cocaine for all known Dnmt’s and methylbinding domain proteins. Among these genes, we found that Dnmt3a is
selectively upregulated at 4 hr and downregulated after 24 hr following both
acute and chronic cocaine administration, with limited changes observed for the
other genes analyzed (Fig. 2-1a, Supplemental Fig. S2-1). Dnmt3a’s
upregulation during early withdrawal time points is supported by equivalent
findings from a recently published microarray study (see supplemental gene lists)
performed 4 hr after 15 chronic cocaine injections (Heiman et al., 2008).
Dnmt3a’s downregulation at 24 hr is not persistent, since mRNA analysis at 48 hr
after the last chronic injection showed no change from control values (data not
shown). Together, these data support the notion that Dnmt3a expression in the
NAc is biphasically regulated in response to each cocaine injection. This biphasic
regulation of Dnmt3a appears to occur via transcriptional mechanisms, since
chromatin immunoprecipitation (ChIP) analysis of a well known marker of gene
activation, histone 3 tri-methyl-Lys4 (me3K4H3), indicated that the Dnmt3a gene,
and not the Dnmt1 or Dnmt3b gene, exhibits transiently enhanced (at 4 hr) and
suppressed (at 24 hr) me3K4H3 binding at its promoter (Fig. 2-1c). To provide
further relevance to addiction, we analyzed tissue from rats that chronically (13
51
days) self-administered cocaine and found a significant downregulation in
Dnmt3a expression in NAc at 24 hr (P<0.05) after the last cocaine infusion (Fig.
2-1b). Interestingly, Dnmt1 is significantly downregulated by acute cocaine
(P<0.05); however this effect was no longer significant with chronic cocaine or
self-administered cocaine (Fig. 2-1a, b). Dnmt3b was not regulated under any
condition analyzed. Moreover, analysis of relative levels of each DNA
methyltransferase in mouse and rat NAc revealed that Dnmt3a is by far the most
predominant DNA methyltransferase expressed in the NAc (Supplemental Fig.
S2-2). Given Dnmt3a’s dynamic regulation by cocaine, and its enrichment in the
NAc, we focused further on this enzyme subtype.
While the molecular events that occur during the transition to the addicted
state, such as the aforementioned biphasic regulation of Dnmt3a, are an
important component underlying the pathophysiology of addiction, a second
major area of research focuses on the mechanisms that maintain the addicted
state. As stated in the Introduction, more long-term regulation of DNA
methyltransferases is of particular interest given the theoretical, persisting
influence that these enzymes may have on downstream gene targets and
behavior. Therefore, we injected mice with cocaine for a more prolonged time
period (28 days) and analyzed Dnmt mRNA levels after an additional 28 days of
withdrawal. This injection paradigm causes robust and long-lasting molecular and
cellular changes, such as increased dendritic spine density on NAc neurons (Lee
et al., 2006; Pulipparacharuvil et al., 2008; Robinson and Kolb, 1999).
Surprisingly, under these conditions, we found that Dnmt3a mRNA expression in
52
the NAc is selectively increased (Fig. 2-2a). We also found that Dnmt3a mRNA
levels are similarly increased in the NAc of rats that underwent 3 weeks of
cocaine self-administration followed by 28 days of withdrawal (Fig. 2-2b).
Together, these mRNA data indicate that Dnmt3a is induced in a sustained
manner after relatively long periods of cocaine withdrawal, and may thereby play
an important role not only in the transition to addiction, but also in the
maintenance of the addicted state.
DNA methylation modulates behavioral responses to cocaine
To understand the behavioral significance of the dynamic regulation of
Dnmt3a in the NAc by cocaine, we utilized complementary pharmacological and
genetic tools to manipulate DNA methylation in this brain region in the context of
cocaine conditioned place preference (CPP), which provides an indirect measure
of cocaine reward. We first administered methionine (a methyl donor)
systemically with an injection regimen reported to hypermethylate ~5% of gene
promoters in brain (Dong et al., 2008) and found that this manipulation caused a
robust decrease in cocaine CPP (Fig. 2-3a). A key limitation of this experiment is
its lack of anatomical and biochemical specificity. Therefore, we next tested
whether intra-NAc delivery of RG108, a potent, highly specific, and nonnucleoside inhibitor of DNA methylation (Brueckner et al., 2005), influences
reward behavior. Indeed, at a dose that decreased global DNA methylation, the
continuous intra-NAc infusion of RG108 markedly enhanced cocaine CPP as well
as the induction of locomotor sensitization to the drug (Fig. 2-3b-d).
53
These data suggest that DNA methylation in the NAc, possibly via
Dnmt3a, negatively regulates cocaine reward. To further test this possibility, we
developed a Herpes Simplex Virus (HSV) vector to temporally and specifically
overexpress Dnmt3a in the NAc (Fig. 2-3e). Dnmt3a overexpression increased
global DNA methylation in this brain region (Fig. 2-3f) and, consistent with
methionine administration, attenuated cocaine CPP (Fig. 2-3g). To obtain the
converse information, we administered an Adeno-Associated Virus (AAV) vector
that expresses Cre recombinase into the NAc of mice homozygous for a floxed
Dnmt3a gene. Such NAc specific knock out of Dnmt3a potentiated cocaine CPP,
an effect not seen for Dnmt1 (Fig. 2-3h). Importantly, none of these
manipulations of DNA methylation in NAc altered baseline locomotor behavior
nor did Dnmt3a overexpression impair general tests of learning and memory
(Supplemental Fig. S2-3). These behavioral data, coupled with the dynamic
regulation of Dnmt3a expression, suggest that increased Dnmt3a expression in
the NAc negatively regulates cocaine reward, whereas decreased Dnmt3a
enhances cocaine reward.
DNA methylation regulates dendritic spine density in NAc
Among the most persistent drug-induced neuroadaptations known is
cocaine’s ability to increase dendritic spine density of NAc neurons (Robinson
and Kolb, 1999). However, the contribution of spinogenesis to the addicted state
remains unclear (Russo et al., 2010) (see Discussion). To gain insight into this
issue, we analyzed NAc neuron spine density in mice that received intra-NAc
54
HSV-Dnmt3a or a control virus after chronic cocaine or saline treatment. Due to
the limited time course of HSV overexpression (which wanes within 6 days of
injection), we used a “5 injection” chronic cocaine schedule which has been
shown to increase spine density 4 hr after the last injection (Maze et al., 2010;
Russo et al., 2009). As expected, cocaine increased NAc spine density and,
interestingly, Dnmt3a overexpression alone was sufficient to increase spine
density to cocaine-comparable levels (Fig. 2-4a).
We next performed a more detailed spine analysis under identical
conditions where we found Dnmt3a expression to be cocaine regulated: at 4 and
24 hr following the last of 7 daily cocaine injections (Fig. 2-1a). Here, we imaged
Lucifer yellow filled NAc medium spiny neurons and analyzed dendrites using
NeuronStudio software; these techniques are amenable to spine density as well
as spine type analysis (Rodriguez et al., 2008). We found an increased spine
density at 4 hr of withdrawal from chronic cocaine (P<0.05) and at 24 hr (P<0.01)
(Fig. 2-4b). Moreover, spine type analysis revealed that both of these effects are
due to a selective increase in thin spines, with no effect seen in mushroom or
stubby spines (Fig. 2-4c). The shared increase in thin spine density suggests a
mechanistic link for these two time points, which might reflect a lasting
consequence of the transient induction of Dnmt3a seen at 4 hr. To test this
hypothesis, we followed the same chronic cocaine dosing regimen while
continuously delivering the Dnmt inhibitor, RG108, into the NAc and analyzed
spine density and spine type 24 hr after the last cocaine dose. We found that
RG108 completely blocks cocaine-induced spinogenesis (Fig. 2-4c). Moreover,
55
spine type analysis revealed that this effect is due to a specific effect on thin
spines (Fig. 2-4c).
DNA methylation in NAc regulates depression-like behavior
In addition to playing an important role in addictive behavior, the NAc is
known to be critically involved in depression and, as with cocaine models, labile
histone modifications such as acetylation and methylation in NAc have been
implicated in rodent models of depression (Covington et al., 2009; Renthal et al.,
2007; Wilkinson et al., 2009). However, the role of DNA methylation in
depressive behavior remains unexplored. We therefore analyzed mRNA levels
for Dnmt’s and methyl-binding domain proteins in NAc at 1 and 10 days following
chronic social defeat stress—an ethologically-relevant model of depression that
induces several depressive-like behaviors including prolonged social avoidance
(Krishnan et al., 2007). We found increased Dnmt3a levels in the NAc at both
time points after chronic defeat stress (Fig. 2-5a). In contrast, no regulation was
seen for any of the other proteins studied (Supplemental Fig. S2-4). As well, no
significant regulation of Dnmt3a mRNA was observed 90 min after the last defeat
nor 2 and 24 hrs after an acute defeat (data not shown).
We next tested the influence of Dnmt3a on susceptibility to social defeat
by subjecting mice, which received HSV-GFP or HSV-Dnmt3a injections into the
NAc, to submaximal defeat stress. In this paradigm, animals undergo just one
day of defeat, which is not sufficient to induce social avoidance; in fact, normal
mice display increased interaction with a social target under these conditions
56
(Krishnan et al., 2007; Krishnan et al., 2008). As expected, HSV-GFP mice
exhibit this significantly increased interaction time (P<0.05). In contrast, Dnmt3a
overexpression in the NAc attenuates social interaction, consistent with a prodepressive-like phenotype (Fig. 2-5b). To complement these data, we assessed
a second model of depressive-like behavior (Porsolt et al., 1977) and found that
Dnmt3a overexpression significantly reduces the latency to immobility (P<0.001)
in the rat forced swim test (Fig. 2-5c), also a pro-depression-like effect. These
data suggest that the prolonged induction of Dnmt3a in NAc by chronic social
defeat stress promotes depressive behavior. To further test this hypothesis, we
continuously infused the Dnmt inhibitor RG108 into the NAc between 1 and 10
days after defeat stress, when we observed increased Dnmt3a mRNA
expression in this brain region.
We found that such local RG108 infusion
reversed social avoidance in defeated mice, an effect similar to that observed by
the standard antidepressant fluoxetine (Fig. 2-5d), indicating that Dnmt inhibition
in this brain region exerts antidepressant-like effects.
57
DISCUSSION
Results of the present study demonstrate that Dnmt3a expression is
subject to dynamic regulation in NAc by two types of chronic emotional stimuli.
Chronic defeat stress induces a persistent upregulation of Dnmt3a in this brain
region. In contrast, cocaine biphasically regulates Dnmt3a expression on a short
timescale, whereas with longer-term withdrawal persisting upregulation of
Dnmt3a is observed as well. Functional experiments establish that the cocaineinduced downregulation of Dnmt3a enhances cocaine reward, whereas
upregulation of Dnmt3a exerts the opposite effect.
Likewise, functional
experiments in the chronic social defeat stress and forced swim paradigms
suggest that prolonged upregulation of Dnmt3a in NAc drives depressive-like
behavior. Taken together, these data suggest that an appropriate balance of
DNA methylation in NAc crucially gates behavioral responses to emotional
stimuli—a hypermethylated state dampens responses to rewarding stimuli and
heightens responses to aversive stimuli, conversely, a hypomethylated state
heightens responses to rewarding stimuli and dampens responses to aversive
stimuli.
Our analysis of dendritic spine density on NAc neurons demonstrates that
DNA methylation is an essential mediator of cocaine-induced spinogenesis:
Dnmt3a overexpression in NAc mimics the cocaine-induced increase in spines,
while RG108 infusion into this region blocks cocaine’s action. A key question is
how such a role for Dnmt3a in NAc spine regulation relates to the highly dynamic
58
regulation seen for Dnmt3a expression. Since Dnmt3a overexpression alone is
sufficient to regulate spine density, and because DNA demethylation is an
enzymatically unfavorable chemical reaction and debate still exists regarding the
enzymatic basis of active DNA demethylation, we speculate that the transient
reduction in Dnmt3a expression, such as what we observe 24 hr after chronic
cocaine, is likely not of a sufficient timescale to downregulate spines (Ooi and
Bestor, 2008). Rather, we speculate that the transient increase in Dnmt3a
expression seen 4 hr after each cocaine exposure leads to the progressive
accumulation of DNA methylation and is thereby responsible for the overall
induction of NAc dendritic spines. The fact that a highly persistent increase in
Dnmt3a expression is seen at 4 weeks of withdrawal, a time point when NAc
spine density is robustly induced as revealed by multiple laboratories and
methods (Lee et al., 2006; Pulipparacharuvil et al., 2008; Robinson and Kolb,
1999), is consistent with our hypothesis. The mechanisms responsible for the
complex time course of Dnmt3a regulation in NAc–with early induction, quickly
followed by suppression, and then followed by a slowly developing but very
sustained induction–is unknown and requires future exploration (see below).
Likewise, the persistent induction of Dnmt3a in NAc after chronic social defeat
stress raises the novel possibility that NAc spine density may be regulated under
these conditions as well, something in fact observed in preliminary investigations
(Christoffell et al., 2010).
Our findings that Dnmt3a induction increases NAc spine density, while it
attenuates cocaine reward, highlights an important question in the addiction field:
59
What is the behavioral relevance of cocaine’s induction of dendritic spines on
NAc neurons? Several conflicting reports exist on this subject (Russo et al.,
2010). Robinson and colleagues positively correlated spine induction with
increased locomotor sensitization (Yao et al., 2004). Three studies that directly
manipulated genes (ΔFosB, NFκB, or G9a) in the NAc, known to regulate spines,
found that manipulations that block spine induction also block cocaine’s
behavioral responses (Maze et al., 2010; Russo et al., 2009). However, two other
studies yielded conflicting results: blocking CDK5 or activating MEF2 blocks
cocaine-induced spinogenesis but enhances cocaine reward (Norrholm et al.,
2003; Pulipparacharuvil et al., 2008). The pattern seen for Dnmt3a matches
these latter findings. The basis for these paradoxical results is unknown. One
possibility is that regulation of different types of spines might exert very different
functional effects on NAc neurons and consequently on behavior. A recent study
reported highly complex regulation of various spine types over a course of
chronic cocaine exposure and withdrawal (Shen et al., 2009), and we show a
selective effect of DNA methylation on the regulation of thin spins. The
observations that Dnmt3a induces thin spines, but blunts cocaine’s behavioral
effects, raise the possibility that the induction of thin spines actually represents a
homeostatic adaptation that serves to oppose the behavioral effects of cocaine.
Clearly, this question requires much further investigation.
The electrophysiological function of cocaine-induced, DNA methylationdependent thin spines remains speculative at this point, however, interesting
electrophysiological correlates have been reported under similar cocaine
60
injection regimens. In association with the increase in thin spines, chronic
cocaine causes: 1) a reduction in firing rate in the NAc shell (Kourrich and
Thomas, 2009), 2) synaptic depression (decreased AMPA/NMDA ratio) during
early (24 hrs) withdrawal (Kourrich et al., 2007), and 3) synaptic potentiation
(increased AMPA/NMDA ratio) during late (10-14 days) withdrawal (Kourrich et
al., 2007). First, some evidence exists for spines driving neuronal firing rate, as it
was recently found that the spine density reductions that are associated with
dopamine depletion induce a homeostatic response of increased firing of medium
spiny neurons (Azdad et al., 2009). Second, synaptic depression, indicated by
decreased AMPA/NMDA ratio, may represent an increased pool of AMPA
receptor-lacking silent synapses, which are also known to be regulated by
cocaine (Huang et al., 2009). Since thin spines represent highly plastic, newly
formed spines which can lack AMPA receptors, we speculate that cocaineinduced DNA methylation induces thin spines that form silent synapses (Kasai et
al., 2010). Finally, the influence of DNA methylation on synaptic plasticity is
heavily supported in the hippocampal literature. In cultured hippocampal
neurons, consistent with our discovery that RG108 reduces cocaine-induced
spine density of NAc neurons, Dnmt inhibition causes a marked reduction in
functional synapses in an activity-dependent manner as measured by a reduction
in mEPSC frequency (Nelson et al., 2008). Moreover, both Dnmt inhibition and
conditional knock out of Dnmt1 and Dnmt3a block long-term potentiation, a
process thought to involve the formation and/or consolidation of dendritic spines
(Feng et al., 2010; Miller and Sweatt, 2007). These studies point to the
61
importance of understanding the electrophysiological changes brought about by
DNA methylation in the NAc. Such studies will provide key insight into whether
cocaine-induced, DNA methylation-dependent changes in thin spines are a
cause or consequence of changes in firing rate and/or synaptic depression.
As noted earlier, the mechanisms responsible for cocaine’s short-term
biphasic regulation of Dnmt3a mRNA expression are unknown. In hippocampus,
Dnmt3a was also found to be rapidly upregulated by fear conditioning, but the
expression pattern following this upregulation over longer time points has not
been assessed. Since Dnmt inhibition in hippocampus blocks memory formation,
it was presumed in this study that Dnmt3a’s transient increase initiates
downstream methylation of target genes that ultimately influence fear memory
(Miller and Sweatt, 2007). This is analogous to our proposal that the transient
increases in Dnmt3a that occur with each cocaine injection cause more lasting
regulation of cellular and behavioral plasticity. In our study, the fact that
me3K4H3 binding to the Dnmt3a promoter is commensurately associated with
Dnmt3a mRNA regulation at both the 4 and 24 hr time points suggests that a
histone 3, Lys4 methyltransferase, such as KMT2A (also known as MLL1), may
regulate Dnmt3a’s expression. Additionally, me3K4H3 is well known to inversely
correlate with DNA methylation at particular gene promoters, raising the
intriguing possibility that Dnmt3a may feed back and regulate its own mRNA
expression (Weber et al., 2007). On a general level, one hypothesis that may
explain Dnmt3a’s complex regulation could be a transcriptional response to the
rapid fluctuations in levels of dopamine or BDNF that are seen with cocaine
62
exposures (Graham et al., 2007). This is an intriguing possibility since 1) BDNF
protein, like Dnmt3a mRNA, also accumulates with prolonged cocaine
withdrawal, and 2) chronic social defeat stress induces lasting enhancement of
BDNF signaling in the NAc (Berton et al., 2006; Krishnan et al., 2007) and, as we
show here, also causes lasting increases Dnmt3a expression in this brain region.
Finally, these data, in conjunction with studies of histone acetylation and
methylation (Covington et al., 2009; Maze et al., 2010; Renthal et al., 2007;
Renthal et al., 2009; Wilkinson et al., 2009), support an emerging model that
epigenetic alterations in the NAc have profound effects on the regulation of
emotional behavior in models of both drug addiction and depression. The
regulation is complex, with different epigenetic mechanisms apparently exerting
distinct effects on behavioral endpoints. A promising line of future research would
be to assess the behavioral function of additional enzymes that mediate still other
forms of epigenetic modifications. In parallel, the gene targets (and their degree
of overlap) for these various enzymes remain largely unknown. The elaboration
of these targets should help us identify the many ways in which epigenetic
mechanisms, including Dnmt3a-mediated DNA methylation, regulate NAc
neuronal function to mediate the complex behavioral phenotypes of addiction and
depression.
In this study, we found that both cocaine and chronic stress differentially
regulate the de-novo DNA methyltransferase, Dnmt3a, in the NAc to control
dendritic spine plasticity and behavioral responses to cocaine and stress. We find
that Dnmt3a activity in the NAc in vivo is necessary for cocaine-induced
63
increases in dendritic spine density selectively for the thin type of spines. Our
findings also suggest that the rewarding responses to cocaine negatively
correlate with thin spines and also raise the possibility that depressive responses
to chronic stress may positively correlate with increased spine density. Taken
together, these observations implicate a new epigenetic modification–DNA
methylation–in the molecular mechanisms controlling cocaine- and stressinduced structural and behavioral plasticity and could ultimately lead to the
development of improved treatments for drug addiction and depression.
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CHAPTER 2 FIGURES
65
Fig. 2-1. Transcriptional regulation of Dnmt3a by chronic cocaine. (a) qPCR
analysis of NAc of acute and chronic (7 days) cocaine treated mice (20
mg/kg/day IP) demonstrated transient increases in Dnmt3a mRNA levels 4 hr
after the final injection (*P<0.05, n=14 acute, n=7 chronic) and a decrease after
24 hr (**P<0.005, n=6 acute, n=16 chronic). Dnmt1 (#P=0.08) and Dnmt3b
transcripts were not altered significantly by chronic cocaine, however, acute
cocaine significantly reduced Dnmt1 expression at 24 hr (*P<0.05, n=6). (b)
Dnmt3a mRNA was significantly reduced in the NAc of chronic (13 days) selfadministering rats examined 24 hr after the last drug dose (*P<0.05, #P=0.12,
n=6 control, n=7 self-administration). (c) ChIP analysis revealed a significant
cocaine-induced increase (4 hr) and decrease (24 hr) in me3K4H3 binding -500
bp upstream to the Dnmt3a promoter (*P<0.05, n=4, 5 mice pooled/n), with no
regulation seen -2000 bp upstream from the promoter.
66
67
Fig. 2-2. Prolonged induction of Dnmt3a by chronic cocaine after 28 days of
withdrawal. (a) qPCR analysis of NAc of chronic (28 days) cocaine treated mice
(20 mg/kg/day IP) that have undergone 28 days of drug withdrawal demonstrated
an increase in Dnmt3a mRNA levels (*P<0.05, n=11). (b) Dnmt3a mRNA
expression was significantly increased after 28 days of withdrawal in the NAc of
chronic (3 week) self-administering rats (*P<0.05, n=6 control, n=7 selfadministration).
68
69
Fig. 2-3. DNA methylation regulates cocaine reward. (a) Chronic (7 days)
methionine (0.78 g/kg/2x/day SC) diminished the rewarding effects of cocaine in
the CPP paradigm (*P<0.05, n=9). (b) Continuous intra-NAc infusion over 7 days
of the DNMT inhibitor RG108 (100 μm) decreased global DNA methylation levels
in NAc (*P<0.005, n=6 vehicle, n=7 RG108), (c) increased cocaine CPP at 10
mg/kg IP (**P<0.0005, n=9), (d) and enhanced the induction of locomotor
sensitization to chronic cocaine (20 mg/kg IP) (*P<0.05, n=8). (e) Verification of
anatomical placement and viral infection in NAc after HSV-Dnmt3a-GFP
injection; immunostaining for GFP is shown. Cartoon adapted from MBSC atlas,
Figure 18, 1.54 mm Bregma. (f) HSV-Dnmt3a increased global DNA methylation
levels (*P<0.05, n=8 HSV-GFP, n=5 HSV-Dnmt3a). (g) Intra-NAc HSV-Dnmt3a
significantly attenuated cocaine reward at 10 mg/kg cocaine (*P<0.05, n=10),
with a trend seen at a lower dose (#P=0.13, n=10). (h) Intra-NAc AAV-Cre
injected into floxed-Dnmt3a mice significantly increased cocaine CPP at 7.5
mg/kg (*P<0.05, n=14 AAV-CRE, n=18 AAV-GFP), with no effect seen in floxed
Dnmt1 mice. All raw CPP data are provided in Supplemental Table 2-1.
70
71
Fig. 2-4. DNA methylation regulates NAc dendritic spine density. (a) Dnmt3a
overexpression mimicked cocaine’s ability to increase dendritic spine density of
NAc medium spiny neurons compared to saline GFP controls (*P<0.05, n=5 both
HSV-GFP conditions, n=6 both HSV-Dnmt3a conditions). Representative
confocal scans of GFP immunostained NAc neurons infected with either GFP or
Dnmt3a-GFP viruses. To correspond with the shortened timescale of HSV
expression, these analyses were performed 4 hr after 5 cocaine injections
(20mg/kg IP). (b) Using an alternate method involving direct injection of Lucifer
yellow into identified neurons, we found that chronic (7 days) cocaine treatment
also significantly increased spine density at 4 hr (*P<0.05, n=7 saline, n=8
cocaine) and 24 hr (**P<0.01, n=8 saline, n=7 cocaine) after the last cocaine
injection. Continuous (7 days) intra-NAc RG108 infusion significantly reduced
spine density 24 hr after the last chronic cocaine injection (*P<0.05, n=4 Vehicle
infused, n=5 RG108 infused). Data are expressed as % change in spine density
relative to saline controls. (c) NeuronStudio spine type analysis of the same
images from (b) revealed that chronic cocaine selectively increased thin spines
both 4 and 24 hr after the last cocaine injection (*P<0.05, **P<0.01). Likewise,
intra-NAc RG108 selectively reduced thin spines of chronic cocaine injected
animals (*P<0.05). None of these conditions significantly affected the number of
mushroom (mshrm) or stubby (stb) spines.
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73
Fig. 2-5. Regulation of depression-like behavior by Dnmt3a. (a) qPCR of NAc
taken 1 or 10 days after chronic (10 days) social defeat shows a significant
increase in Dnmt3a levels at both time points (*P<0.05). Inset displays social
avoidance, a depressive-like behavior, observed in these mice (**P<0.005, n=16
control, 26 defeated). (b) Naïve mice infused intra-NAc with HSV-GFP or HSVDnmt3a were subjected to a submaximal protocol of social defeat. HSV-GFP
mice showed a significant increase in interaction with a social target (F1,42 =
5.219, *P<0.05) as would be expected from control mice25, whereas HSVDnmt3a mice lacked this phenotype (P>0.05), a pro-depressive-like response. (c)
Intra-NAc injection of HSV-Dnmt3a significantly decreased latency to immobility
on Day 2 of the rat forced-swim test, also a pro-depressive-like response
(**P<0.001 n=7). (d) Conversely, intra-NAc infusion (10 days) of RG108, initiated
one day after the last defeat episode, completely reversed the chronic social
defeat-induced social avoidance exhibited by vehicle-infused mice (F1,44 = 12.876
**P<0.001), an effect equivalent to that seen for chronic (20 mg/kg/day IP, 14
days) fluoxetine administration (P>0.05).
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CHAPTER 2 SUPPLEMENTAL FIGURES
75
Supplemental Figure S2-1. mRNA profiling of methyl-binding domain
proteins (Mbd’s) after chronic cocaine. (a) qPCR analysis of NAc of chronic (7
days) cocaine treated mice indicated lack of regulation of Mbd’s at 4 and 24 hrs,
with the exception of a significant increase in Mbd3 at 4 hrs (*P<0.05, n=7) and
Mbd1 at 24 hrs (*P<0.05, n=16). (b) However, Mbd1 and other Mbd’s were
unaltered in the NAc of self-administering rats examined 24 hours after the last
cocaine dose.
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Supplemental Figure S2-2. Relative Dnmt mRNA levels in NAc of mice and
rats. Data expressed as fold difference from the average level of all Dnmt’s from
20 control mice and 6 control rats. Dnmt3a shows the highest expression in the
NAc. Dnmt expression was measure by qPCR.
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78
Supplemental Figure S2-3. Experimental manipulation of DNA methylation
does not alter locomotor activity or learning. (a) Locomotor activity was
measured by photobeam breaks in the CPP chamber during the first day of
saline training. We found no significant locomotor effects of subcutaneous
methionine, intra-NAc RG108, or intra-NAc HSV-Dnmt3a. (b) Mice given intraNAc injections of HSV-GFP or HSV-Dnmt3a spend equally more time with a
novel, substituted object compared to non-substituted objects. Mice were
repeatedly (3 times, 5 min each) exposed to 3 objects with 3 min intervals
between test sessions. On the final trial, one object is substituted with a novel
object. (c) HSV-GFP and HSV-Dnmt3a treated mice become equally
disinterested in a single novel object upon reexposure. Mice were repeatedly
exposed to a single object for 5 min with 1 hr intervals between test sessions.
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Supplemental Figure S2-4. NAc mRNA profiling of Dnmt1, Mbd1, and Mbd4
following social defeat. (a) qPCR analysis of NAc of mice subjected to chronic
(10 days) social defeat taken 1 or 10 days after social interaction tests (Day 12
and 21) indicated lack of significant regulation of Dnmt1, Mbd1, and Mbd4
(P>0.05).
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Supplemental Table 2-1. Raw CPP data from figure 2-3 (a-h).
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CHAPTER 3
GENOME WIDE ANALYSIS OF COCAINE-INDUCED DNA METHYLATION
ABSTRACT
Studies in Chapter 2 establish that DNA methylation plays a key role in
regulating drug reward and synaptic plasticity. The next major challenge lies in
identifying the molecular mechanism of how DNA methylation exerts these
effects. Thus, I optimized a technique called mDIP (methylated DNA
Immunoprecipitation) for analyzing in vivo NAc DNA methylation levels across all
gene promoters in the genome. Ultimately, these data point to a lack of
detectable cocaine-induced regulation in gene promoters. However, a gene
expression screen for alterations that occur in response to NAc Dnmt3a
overexpression revealed that immediate early genes (IEG’s) are specifically
silenced by this alteration. Conversely, Dnmt inhibition robustly upregulates the
expression of a large subset of genes that are also regulated in an activitydependent manner. In addition to neuronal activity, IEG’s are well-known to be
upregulated by cocaine, however, the epigenetic mechanisms that mediate their
return to basal levels are unclear. My discovery that DNA methylation selectively
regulates IEG expression raises the possibility that DNA methylation may be
involved in regulating cocaine induced IEG repression. Based on the mDIP-Chip
data, the lack of differential DNA methylation at IEG gene promoter suggests that
this regulation may be transient, occurring in a minority of cells, and/or occurring
in genomic regions distal to IEG gene promoters.
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INTRODUCTION
A hallmark of virtually all known mechanisms of long-lived neuronal
plasticity is the dependence on transient intracellular signaling cascades.
Thanks to major advances in biochemistry, over the past several decades
neuroscientists have unraveled many mysteries of how neurons accomplish
changes in synaptic function. On a molecular scale, key examples include
insertion/removal of receptor subunits from synaptic membranes, alteration of
synaptic protein function via posttranslational modifications, and the stimulation
of the translation or degradation of proteins at the synapse. However, all these
mechanisms occur at the protein level and are therefore subject to eventual
turnover. Neuroscientists have long ignored the fact that the only permanent
structure in any given neuron is its genome. Therefore, these transient
intracellular signaling cascades that initiate all forms of neuronal plasticity must in
some way be recorded in the genome. In order to adapt properly, each neuron
must retain a “memory” of where it has been—what genes were expressed, and
just as importantly what genes were not.
Because it is long-lived, methylation of DNA is a very likely candidate to
assist neurons in achieving these putative ‘genomic records.’ In the past 5 years,
several key studies have now implicated DNA methylation in regulating synaptic
plasticity. For example, DNA methylation regulates activity-dependent functional
synapses, long term potentiation (LTP), long term depression (LTD), as well as
the formation of dendritic spines (Feng et al., 2010; LaPlant, 2009; Levenson et
83
al., 2006; Miller and Sweatt, 2007). Moreover, DNA methylation is implicated in
playing a major role in several neurological diseases: autism, mental retardation,
schizophrenia, addiction, depression, and disorders of memory formation. In
parallel with these discoveries, over the past few years molecular and genomic
techniques have been developed that allow one to analyze DNA methylation
across all genes in the entire genome. In fact, one technique, mDIP-Chip
(methylated DNA Immunoprecipitation on chip) has led to the identification of
hundreds of gene promoters that are hypermethylated in cancer cells (Weber et
al., 2007). And just two months ago (March 2010), an mDIP-chip study
performed on hippocampal tissue from Dnmt1/3a double knock out mice
identified 161 hypomethylated gene promoters (Feng et al., 2010).
The key next step will be to identify DNA methylation of genes that occur
in wild type neurons in response to transient stimuli that are known to alter
neuronal plasticity and behavior in a DNA methylation-dependent manner. To
identify such genes, I employed a mouse chronic cocaine administration model. I
focus on this model because I have found that DNA methylation plays an
essential role in regulating dendritic spine homeostasis as well as behavioral
responses to cocaine.
The specific goal of this work is to identify DNA methylation changes that
occur in the mouse NAc genome in response to chronic cocaine. I used 3
approaches. First, I performed an mDIP-Chip experiment for analyzing DNA
methylation in all promoters in the genome. To correlate putative methylation
changes with changes in gene expression, I performed two gene expression
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array experiments after chronic cocaine. In a second approach, I overexpressed
Dnmt3a in NAc tissue and performed a gene expression screen of 100 genes.
Third, I preformed a gene expression array after Dnmt inhibition of cultured
neurons in vitro.
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METHODS
Animals
We used adult male c57bl6j mice (Jackson laboratory) and Long-Evans
rats (Charles River; self administration experiment). For all experiments, animals
were habituated in our facility 1 week prior to experimentation and were housed
on a 12 hr light-dark cycle with access to food and water ad libitum. All animal
experiments were approved by the IACUC’s of Mount Sinai School of Medicine
and UT Southwestern.
Drugs
For chronic cocaine (Sigma) experiments, we used a standard cocaine
injection which includes 7 daily intraperitoneal (IP) injections of 20 mg/kg
cocaine. Control mice were injected with an equivalent volume of saline.
Striatal cultures
Embyronic striatal neurons (E18) were cultured from Long Evans rats
(Charles river labs) as described previously without modifications
(Pulipparacharuvil et al., 2008). The cells were kept for 10 days in two 6 well
dishes with 8 x 106 cell/ well (12 total wells for 4 conditions ran in triplicate). On
the 11th day, 100 μm RG108 (in 0.2% DMSO) or DMSO (0.2%) was applied.
After 24 hours, half of the wells were bathed in depolarizing solution (60 mM KCl)
for 1 hour. RNA from each well was immediately harvested for microarray
86
analysis. Cells were monitored on a microscope daily to during the timecourse of
the experiment to ensure that they remained healthy throughout the experiment.
Neither RG108 nor KCl caused any overt change in cell number or morphology
as observed by eye at high magnification.
Herpes simplex virus injections
We used the bi-cistronic p1005+ HSV vector, which expressed GFP alone
or GFP with Dnmt3a. In this system, HSV infection occurs selectively in neurons.
GFP expression is driven under the human immediate early cytomegalovirus
(CMV) promoter, while the gene of interest, Dnmt3a, is driven by the IE4/5
promoter (Clark et al., 2002). The mouse Dnmt3a-1 plasmid (Linhart et al., 2007)
was subcloned into the HSV vector, the vector was sequenced and then
packaged into high-titer viral particles as described previously (Barrot et al.,
2002). Stereotaxic surgery was performed on mice under general anesthesia
with a ketamine/xylazine cocktail. Relative to Bregma, coordinates to target both
the NAc shell and core were: 10° A/P +1.6 mm, M/L +1.5 mm, and D/V -4.4 mm.
We bilaterally infused 0.5 μl at a rate of 0.1 μl/min. Mice were sacrificed 4 days
post-op and 1 mm NAc tissue punches were taken after GFP visualization on a
Leica fluorescent microscope.
DNA isolation
Frozen tissue punches were homogenized with a handheld pipette in 150
μL lysis buffer (20 mM Tris-HCL, 1mM EDTA, 400 mM NaCl, 1% SDS,
87
Proteinase K) and incubated for 3 hours at 65 degrees. Then 2 μL RNase A
(Fisher Scientific) (100 mg/ml) was added and kept for 30 minutes at 25 degrees.
One volume of Phenol:Chloroform:Isoamyl-Alcohol was added and briefly shaken
in phase lock tubes (Qiagen Maxstract). Phase lock tubes were then centrifuged
at 12,000 rmp for 10 min and aqueous phase was collected. Then standard
ethanol precipitation is carried out with 100% and 70% ethanol using glycogen as
a carrier. The pellet was dried and resuspended in 0.1x TE buffer. DNA amount
and purity was then quantified using Nanodrop (Thermo Scientific).
Methylated DNA immunoprecipitation
i. Sonication of genomic DNA
Genomic DNA is randomly sheared by sonication to generate fragments between
300 and 1000 bp. First, 10-20 μg genomic DNA is diluted in TE in a 1.5 ml
eppendorf tube (10-20 μg DNA in 400 μl TE). Then sonicate 4 times 10 seconds
(BRANSON digital Sonifier model 450, used with the tapered Microtip, amplitude
20%), with 1 minute intervals between pulses (keep the tube on ice during the
sonication). 8 μl was then ran on an agarose gel to check for appropriate
fragmentation. Precipitate the sonicated DNA with 400 mM NaCl, glycogen (1 μl)
and 2 volumes 100% ethanol. Resuspend the DNA pellet in TE and measure
DNA concentration
ii. Immunoprecipitation
Sonicated DNA was immunoprecipitated with a monoclonal antibody
against 5-methylcytidine (5mC) (EUROGENTEC #BI-MECY-1000) or an IGG
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control. A portion of the sonicated DNA is left untreated to serve as input control.
Immunoprecipitation is performed as follows:
1. Dilute 4 μg of sonicated DNA in 450 μl 0.1xTE
2. Denature for 10 minutes in boiling water and immediately cool on ice for 10
minutes
3. remove 45 μL for Input.
4. Add 46 μl of 10x IP buffer (100 mM Na-Phosphate pH 7.0, 1.4 M NaCl, 0.5 %
Triton X-100)
5. Add 2 μl of 5mC antibody or 2 μL IgG control
6. Incubate 2 hours at 4°C with overhead shaking
7. Pre-wash 40 μl of Dynabeads with 800 μl PBS-BSA 0.1% for 5 minutes at RT
with shaking
8. Collect the beads with a magnetic rack and repeat wash with 800 μl PBS-BSA
0.1%
9. Collect the beads with a magnetic rack and resuspend in 40 μl of 1x IP buffer
10. Add Dynabeads to the sample
11. Incubate 2 hours at 4°C with overhead shaking
12. Collect the beads with a magnetic rack and wash with 700 μl 1x IP buffer for
10 minutes at RT with shaking
13. Repeat wash with 700 μl 1x IP buffer twice
14. Collect the beads with a magnetic rack and resuspend in 250 μl proteinase K
digestion buffer
15. Add 7 μl proteinase K (10 mg/ml stock)
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16. Incubate 3 hours at 50°C (use a shaking heating block 800 rpm to prevent
sedimentation of the beads)
17. Prepare phase lock tubes: 14k-30 seconds. Add samples to tubes. Add 1
volume of phenol:chloroform:IAA (250 μl). Shake 30seconds. Spin 14k – 8min
room temp.
18. Precipitate the DNA with 400 mM NaCl (20 μl NaCl 5M), glycogen (1 μl) and
2 volumes 100% ethanol (no more than 500 μl). Keep at -80° for 30 minutes then
centrifuge samples at 14k rpm for 25min.
19. Resuspend the DNA pellet in 30 μl 0.1xTE and keep at –20°C until later use.
iii. mDNA analysis by PCR and Whole Genome Amplification (WGA)
for microarray
Enrichments in the MeDIP fraction were measured by real-time PCR and by
microarray analysis. For real-time PCR, I used 20 ng of total input DNA and 2 μl
of MeDIP DNA per reaction. Enrichments in the MeDIP fraction are calculated
relative to an unmethylated control, CSA, which is an unmethylated CpG island
containing promoter. For genome-wide analyses, input and MeDIP fractions are
differentially amplified using Sigma’s Whole genome amplification kit without
modifications. Labeled with Cy3 and Cy5 and co-hybridized to microarrays as a
two-color experiment. The methylation level is measured as the intensity ratio of
immunoprecipitated to input DNA.
90
iv. mDIP-Chip analysis
Samples were labeled with Cy3 (input) or Cy5 (meDNA enriched) and hybridized
to NimbleGen MM8 mouse “promoter plus” arrays with 3 biological replicates
used per condition. Each replicate consisted of bilateral NAc punches pooled
from 10 mice. ChIP-chip analysis was performed as described previously without
modification (Renthal et al., 2009; Wilkinson et al., 2009). Briefly, pre-processed,
normalized probes are assigned an alteration score which is based on mean
probe intensity relative to input, the difference in cocaine mean intensity
compared to saline, and a moving average of this signal difference. Alteration
scores that were more than 3.1 standard deviations from the mean were
considered significant (p<0.001).
RNA isolation
Bilateral NAc punches were dissected from mice treated with the indicated
experiment and frozen on dry ice. Frozen brain tissue was then homogenized in
TriZol (Invitrogen) and processed according to the manufacturer's protocol. RNA
was purified with RNeasy Micro columns (QIAGEN) and processed as indicated
by the manufacturer. Spectroscopy confirmed that the RNA had 260/280 and
260/230 ratios >1.8. Reverse transcription of total RNA (1.2 μg) was carried out
using iScript (Biorad) as indicated by the manufacturer. The cDNA is then diluted
to 4 ng/μL (assuming 100% efficiency from RT reaction). qPCR was then run in
10 μL triplicate reactions on 384 well plate using approximately 4 ng of cDNA per
reaction, 10 mM primers, and SYBR Green (ABI). Each reaction was quantified
91
using the ΔΔCt method as previously described (Tsankova et al., 2006). Using
the above method for RNA isolation and RT, I have optimized these PCR
conditions to allow for analysis of more than 100 genes from a single mouse
NAc—effectively increasing the number of PCRs by 10 fold compared to
previous work in mouse NAc (Renthal et al., 2007).
cDNA microarray analysis
RNA isolation was performed exactly as described above on bilateral
punches that were pooled from 4 animals per replicate. For the 24 hour
microarray, there were 6 replicates per group (cocaine vs saline) and for the 1
hour microarrays (mouse cocaine, and rat striatal culture); there were 3
replicates per group. Microarray processing and data analysis were performed as
previously described (Covington et al., 2009; Krishnan et al., 2007). Briefly,
purified RNA was checked for quality using Agilent’s Bioanalyzer (Santa Clara,
CA). Reverse transcription, amplification, labeling, and hybridization to Illumina
MouseWG-6 V2.0 or RatWG-12 V2.0 arrays were performed using standard
procedures by UT Southwestern’s microarray core. Raw data were background
subtracted and quantile normalized using Beadstudio software (Illumina, San
Diego, CA). Normalized data were then analyzed using GeneSpring software
(Agilent). The dataset was filtered to only analyze transcripts that were
significantly detected (p<0.01) across all 18 microarray. ‘Significant’ genelists
were generated using this filtered list in addition to significance criteria of 1.3 fold
change cutoff coupled with a non-stringent p-value cutoff of p < 0.05. We have a
92
high degree of confidence in these data because the data analysis criteria used
for our study are recommended by the MicroArray Quality Control (MAQC)
project because these criteria have been validated to have the highest inter-site
reproducibility and intra-platform reproducibility. Second, many genes that are
well known to be cocaine regulated (fos, fosB, fosbl2, junB, egr1, egr2, egr4,
hspa1, npas4, per2) were on these lists. Furthermore, independent PCR analysis
validated differential expression of 12 genes from the 1 hour genelist (Maze et
al., 2010) and 7 genes from the 24 hour genelist (table 3-1).
93
RESULTS
Optimization of methylated DNA IP in brain tissue
As a first step to identifying differential DNA methylation on a genome
wide scale, I optimized methylated DNA immunoprecipitation (mDIP) in mouse
NAc tissue. The greatest hurdle in genome wide studies is the development and
utilization of appropriate statistical analysis. Therefore, mDIP was determined to
be particularly suitable for my studies because this technique would allow for the
usage of the exact same Nimblegen promoter arrays which have been optimized
and developed for data analysis for similar ChIP-chip studies (McClung et al.,
2005; Renthal et al., 2009; Wilkinson et al., 2009). Moreover, this technique was
deemed appropriate since it was successfully used to identify differential
methylation in the genomes of cancer cells compared to healthy cells (Weber et
al., 2005; Weber et al., 2007). However, at the time of study, mDIP had only
been performed on cell lines in vitro. Therefore, it was crucial to reproduce key
basic findings from cell culture studies to mouse NAc tissue homogenates. To
this end, I developed an “in house” mDIP protocol which combines aspects of the
published mDIP method with the Nestler lab’s ChIP protocol (see mDIP protocol,
methods section). Importantly, I optimized sonication conditions which reproduce
the suggested size fractionation range of 300 to 1000 bp (Fig. 3-1a). Second,
using 3 positive control loci (IAP and H19.1 and H19.3) and 2 negative control
genomic loci (β-actin and CSA), I optimized the antibody-to-DNA ratio to
reproduce published enrichment levels of known methylated and unmethylated
94
genomic loci. IAP is a retrotransposible element that exists in approximately 1000
copies per cell and is 100% methylated. H19.1 and H19.3 are two adjacent loci of
an imprinted control region that is also 100% methylated. Finally, β-actin and
CSA have been shown to lack methylation using a variety of techniques (Weber
et al., 2005). As shown in Fig. 3-1b, it turns out that the antibody-to-DNA ratio
dramatically effects PCR enrichment of methylated genes. Higher concentrations
of antibody non-specifically enrich unmethylated DNA, whereas the lowest
concentration of antibody exactly replicated the fold enrichment of positive and
negative controls (Weber et al., 2005; Weber et al., 2007). Moreover, the
standard error in NAc tissue in these experiments is actually considerably lower
than the data from cell culture in the published method (Weber et al., 2005;
Weber et al., 2007). Third, I determined the appropriate conditions for whole
genome amplification (WGA) to produce enough DNA for microarray
hybridization without compromising the relative methylation enrichment of
positive and negative controls (Fig 3-1b). Overall, these data demonstrate that
mDIP-chip is applicable in brain tissue for analyzing differences in DNA
methylation for up to two orders of magnitude. Finally, before carrying out the
cocaine mDIP-chip experiment, I performed a fourth “proof-of-principle”
experiment to demonstrate that it is possible to detect differences in brain DNA
methylation. Here, based on published differences in the striatum (STr)
compared to the olfactory bulb (Ob) of the mRNA expression of tyrosine
hydroxylase (TH), dopamine receptor 1a (Drd1a), and the shaker-related voltage
gated potassium channel (Kcna4), I analyzed methylation of these three genes
95
across these brain structures (Fig. 3-1c) (Lein et al., 2007). Despite having very
high expression in the Ob and low expression in the STr, TH was heavily
enriched (~20 fold greater than actin) in both brain regions. However, TH had
significantly higher methylation in the STr. Conversely, Drd1a had significantly
higher methylation in the Ob and overall had moderate (~5 fold greater than
actin) methylation levels. Consistent with the fact that the majority of genes have
minimal levels of methylation in their promoter regions, I found that kcna4 had
undetectable methylation in both brain regions.
DNA methylation analysis in NAc tissue after chronic cocaine
To identify whether cocaine persistently alters DNA methylation in the
NAc, I performed an mDIP-Chip experiment in mice that were injected with
chronic cocaine (7 days, 20 mg/kg) that had undergone 24 hours of drug
withdrawal. To maximize potential for comparative analysis, this design was used
to match our ongoing ChIP-chip studies at this cocaine withdrawal timepoint
(Renthal et al., 2009). The chips contain 385K probes that span a 2 kb region
across approximately 19,000 gene promoters in the mouse genome. Of note,
one half of the NAc’s from this experiment were used for a cDNA microarray.
Also for cDNA microarray analysis, I sacrificed a second group of mice 1 hour
after the last chronic cocaine injection. The purpose of the two cDNA microarray
studies is to assist in identifying transcriptionally relevant genomic loci.
For quality control analysis, I verified that methylation enrichment occurred
at the expected genomic loci. As predicted, both TH and H19 were in the top 1st
96
percentile of methylated promoters (IAP promoter probes not available), Drd1a
was in the top 20th percentile, and negative controls were in the bottom 30th
percentile. Similarly, if I take the top 20th percentile of methylated genes (3894
genes) and analyze absolute mRNA expression, I find that the majority (>90%) of
these gene promoters are associated with transcripts that are expressed at low
levels (supplemental Fig. S3-1). These findings provide a high degree of
confidence in the technical validity of this mDIP-Chip experiment.
Using standard parameters (Renthal et al., 2009; Wilkinson et al., 2009)
for saline versus cocaine comparative chip analysis, I found that 1711 gene
promoters (approximately 9% of all analyzed gene promoters) showed significant
changes in methylation by chronic cocaine compared to saline treated animals
(Fig 3-2a, p<0.001). Specifically, 1228 gene promoters were hypermethylated
and 484 were hypomethylated. Moreover, analysis of differential gene expression
at 1 hour and 24 hours after chronic cocaine treatment revealed that
approximately 13% and 4.8% of genes significantly regulated at the mRNA level
are also differentially methylated (Fig. 3-2a). However, despite their statistical
significance, the vast majority of promoters exhibiting differential DNA
methylation have small fold change differences compared to saline (generally
ranging from 1.2 to 1.5 fold change). For example, if I further apply a 1.5 fold cutoff to the DNA methylation genelist, I find that only 60 of the 1711 are
differentially methylated (Fig 3-1a). This observation raises concern because
even if such methylation changes do represent biologically significant events, it
will be experimentally challenging to prove that such changes occur.
97
To this end, I developed a candidate genelist of 53 candidate genes. In
order to maximize the possibility of identifying differentially methylated genes, the
genes on this list were carefully selected based on several different criteria. For
example, seven genes in Table 3-1 highlighted in blue were identified at the 24
hr or 1 hr microarray and were independently validated to have differential mRNA
expression. Moreover, ten genes highlighted in orange have been previously
identified as playing an important role in biochemical or behavioral responses to
psychostimulants. Third, I selected 12 of the 60 genes that met the criteria of
reaching both statistical (p<0.001) as well as fold change cutoff (>1.5 fold). In the
field, the gold standard for validation of differential methylation is with bisulfite
sequencing (Shames et al., 2007). Therefore, I utilized a high throughput massspectroscopy based bisulfite sequencing technique (called Sequenom analysis)
(Coolen et al., 2007). The greatest hurdle in Sequenom analysis is in developing
optimal PCR primers and parameters that produce a single pure DNA amplicon
(Coolen et al., 2007). Therefore, as a screen, I designed Sequenom primers that
matched to a 100-500 bp sequence corresponding to the differentially methylated
region of each of these 53 gene promoters and then used the best 26 primer sets
for Seqenom analysis. Unfortunately, even after using an aliquot from the same
DNA that I used for my mDIP-chip experiment, Sequenom analysis of these
genes did not identify significant regulation at any of these 26 loci. Figure 3-3
displays a typical example of methylation analysis one of these genes, Htr3a,
which encodes the serotonin 5HT3a receptor.
98
In-Vivo analysis of DNMT3a gene targets
The mDIP-chip data overwhelmingly point to a lack of differential DNA
methylation occurring at promoters in the genome. However, sizeable evidence
suggests that DNA methylation is functionally relevant to cocaine-induced
behavior in the NAc (LaPlant et al., 2010; see Chapter 2). Dnmt3a, for example,
is transiently upregulated by chronic cocaine and viral-mediated overexpression
of DNMT3a results in significant increase in global DNA methylation levels,
significant increase in dendritic spine density, and a significant decrease in
cocaine conditioned place preference.
Since DNMT3a has such robust effects at a cellular and behavioral level,
an alternative approach to finding cocaine induced methylation targets would be
to see which genes have altered mRNA expression in response Dnmt3a
overexpression. This experiment has an element of added benefit and specificity
since HSV only infects neurons. Therefore, I collected NAc RNA and performed
real-time PCR analysis from mice that were sacrificed during the time of maximal
HSV-Dnmt3a or HSV-GFP overexpression (Table 3-2). Importantly, the majority
of the 100 genes whose expression I profiled are known to play important roles in
drug addition. Moreover, the genes that I selected span a variety of cellular
processes throughout neurons (receptors, ion channels, kinases, neuropeptides,
spine structural genes, chromatin modifying enzymes, and transcription factors).
Overall, 18 genes were significantly regulated by Dnmt3a overexpression (6
99
upregulated, 12 downregulated) (Fig. 3-4). Specific examples of transcripts that
were regulated include: Dicer, a key regulator of miRNA processing, Camk2a
splice variants, Dnmt1, and the spine associated genes, Homer1a, and Psd-95.
Of note, a striking number of regulated genes (10/18) belong to a gene family
that is dynamically regulated by neuronal activity—immediate early genes. In this
family, 83% (10/12 genes) of immediate early genes were downregulated.
Notably, most of these genes, such a c-fos, fosB, and Arc, are well known to be
cocaine regulated (McClung et al., 2004). However, revisiting the mDIP-Chip
DNA methylation binding profiles specifically at these promoters, I observed a
lack of cocaine-induced differential methylation (data not shown)—suggesting
that DNA methylation may regulate cocaine induced gene expression at regions
outside of IEG gene promoters (see discussion). An important experiment that is
underway will be to look at DNA methylation levels at IEG genes after Dnmt3a
overexpression.
DNA methylation regulates activity dependent gene expression
Immediate early genes represent the first wave of transcriptional activation
in response to cocaine. On a basic level, these genes are regulated by a
transient, orchestrated, calcium dependent response to neuronal activity. Since
Dnmt3a overexpression blocks the expression of this gene family, I wanted to
test how inhibiting DNA methylation influences activity dependent gene
expression by depolarizing cultured striatal neurons—a well established model
for assessing activity dependent gene expression (Greer and Greenberg, 2008).
100
Here, striatal neurons were bathed in the Dnmt inhibitor, RG108, or DMSO, for
24 hours and were then subjected to KCl induced depolarization or control
solution. RNA was harvested for microarray analysis immediately after 1 hour of
depolarization. This timepoint was chosen because it is when activity dependent
genes are maximally activated (Greer and Greenberg, 2008). I found that RG108
at baseline significantly increased expression of 95 genes. Heatmap comparison
to how these genes are regulated after depolarization revealed that the majority
of these genes (>65%) are also regulated by KCl (Fig 3-5a). I found that a much
larger number of genes were regulated by neuronal activity alone (131 genes).
Interestingly, heatmap analysis also revealed that many of these genes which
are regulated by activity are also upregulated by RG108 (>40%) (Fig. 3-5a).
Taken together, these data suggest that transcriptional regulation of sizable
subset of activity dependent genes share a functional link with DNA methylation
dependent gene transcription. Surprisingly, however, RG108 in the presence of
KCl does not appear to markedly sensitize (further increase) expression of KCl
regulated genes nor does RG108 sensitize the genome (increase the total
number of genes that are significantly regulated by KCl). Mechanistically, this
suggests either a ceiling effect (genes are maximally regulated by KCl), or
perhaps DNA methylation plays a less of a role in the induction of IEG
transcription and perhaps a more prominent aspect in the repression or
stabilization or IEG expression.
101
DISCUSSION
In summary, chronic cocaine does not cause overtly detectable
differences in DNA methylation at gene promoter regions. This is surprising since
behavioral adaptations to cocaine and cocaine-induced changes in spine density
are mediated at least in part via DNA methylation. Since gene promoters cover
only a small percentage of the genome, it’s plausible that cocaine-induced DNA
methylation may occur outside of promoter regions. To functionally prove that
manipulating DNA methylation does effect gene transcription, I overexpressed
Dnmt3a in the NAc and discovered that Dnmt overexpression results in a marked
and significant decrease in 83% of immediate early genes analyzed. Moreover,
this regulation was selective to immediate early genes, since 82% of transcripts
analyzed were not significantly regulated by Dnmt3a overexpression. In the
converse experiment, in vitro Dnmt inhibition revealed that the vast majority of
genes regulated by DNA methylation are also regulated in an activity-dependent
manner. Therefore future studies point to analyzing NAc DNA methylation levels
following Dnmt3a overexpression, Dnmt inhibition, and cocaine treatment at
genomic regions in and around immediate early genes—particularly at IEG
enhancer elements (discussed below).
The selective role of DNA methylation in IEG expression is of broad
significance in the neuroscience community because IEGs represent the
gateway to the genome’s response to neural activity. This gene family is arguably
the most dynamically regulated of all genes. In response to activity, somehow
neurons are capable of: 1) rapidly activating, 2) rapidly deactivating, and 3)
102
maintaining a silenced, but ready state of IEG gene expression until the next
wave of neuronal activity. On top of these 3 processes, a multitude of examples
exist where re-activation, or re-exposure to the same stimulus, results in
enhanced (so called ‘sensitized’) or attenuated (so called ‘desensitized’) IEG
expression. For example, our group has repeatedly observed that IEG’s such as
c-fos and fosB have desensitized striatal induction in psychostimulant
experienced compared to naïve rodents (Alibhai et al., 2007; Renthal et al.,
2008). Moreover, this desensitized induction persists in animals for a long as 1
month
after
drug
withdrawal
(LaPlant,
2009).
Such
sensitization
or
desensitization has largely been attributed to up-stream post-transcriptional
mechanisms such as differences in phosphorylation, however, the role of DNA
methylation has not been assessed (Edwards et al., 2007). Here, I have
discovered that DNA methylation plays a key role in the regulation of IEG
expression. In addition to identifying if this regulation is directly via DNA
methylation, it will also be important for future research to further understand the
role of DNA methylation in regulating the temporal dynamics of IEG expression.
Similarly, an important line of future research will be to see if repeated neural
activity and IEG activation may result in a type of ‘genomic recording’ via
accumulation of DNA methylation at IEG regulatory elements.
Lack of cocaine-induced DNA methylation at gene promoters
There are three interpretations regarding my meDIP-chip data. 1.
Cocaine-induced DNA methylation is not significantly occurring in the genome. 2.
103
Cocaine-induced DNA methylation is occurring but not in gene promoters. Or 3.
Cocaine-induced DNA methylation occurs in gene promoters, but subtle changes
are at or below the threshold of detection using state-of-the-art techniques.
Based on the 24 hour microarray data, which had cocaine-induced expression
changes typically 1.3-1.5 fold different from saline treated animals, it is not
surprising that subtle changes may be occurring with DNA methylation as well.
An improved approach for future research will be to analyze meDNA and mRNA
in purified neurons or even specific neuronal subtypes. This approach has
resulted in much greater fold differences in mRNA (Heiman et al., 2008). Another
important point that warrants clarification is that bisulfite sequencing may be a
weakness
for
validation
because
it
was
recently
discovered
that
hydoxymethylation of DNA occurs specifically in neurons (Kriaucionis and Heintz,
2009). This caveat ultimately means that sequencing reactions cannot actually
differentiate between methylated cytosine and hydroxymethylated cytosine. At
this point, the extent of hydoxymethylation that occurs throughout the gene
promoter region is not known. An important strength of the mDIP technique is
that the antibody recognizes only meDNA. Therefore, it may be worth revisiting
my validation experiment by using the mDIP technique. To truly answer the 1st
and 2nd possibilities (whether DNA methylation occurs outside of promoter
regions), the next major experiment will need to test DNA methylation levels
throughout the entire genome using sequencing technology. A key weakness of
my study was that the microarray only covered approximately 2 kb for all 19,000
ref-seq gene promoters. While this may sound like a lot, overall this is only about
104
1.5% of the mouse genome. Recently, several high profile whole-genome
sequencing publications have emerged which suggest that DNA methylation is
playing a much bigger role outside of CpG islands than in gene promoters. One
study found that the region distal to CpG islands containing gene promoters-so
called “CpG shores”-is subject to heavy differential DNA methylation (Irizarry et
al., 2009). Of major interest, a histone methylation ChIP-sequencing study done
in neuronal culture found that activity dependent gene expression is heavily
linked to epigenetic regulation at enhancer elements several thousand base pairs
away from the canonical promoter region (Kim et al., 2010). Moreover, this study
found that while me3K4H3 was highly enriched at activity dependent gene
promoter regions, me1K4H3 was highly enriched at enhancer elements of these
genes (Kim et al., 2010). This has important implications for a role of DNA
methylation in activity dependent gene regulation because it is well known that
the level of methylation on K4H3 is inversely correlated with the level of DNA
methylation (Weber et al., 2007). Taken together, these studies highly suggest
that the most likely genomic target of DNA methylation is at IEG enhancer
elements.
Link between Dnmt3a’s influence on gene expression and Dnmt3a’s known
cellular and behavioral role
As previously mentioned, I found that intra-NAc Dnmt3a overexpression
increases dendritic spine density and decreases cocaine CPP. A high degree of
evidence exists which supports the notion that IEGs are critically linked to
105
cocaine reward. IEGs are well known to be induced by cocaine (Hope et al.,
1994). Several studies have found that pharmacologic or genetic attenuation of
IEG induction is correlated with reduced behavioral response to cocaine (Kumar
et al., 2005; Maze et al., 2010). However, one disconnect between cocaine’s and
Dnmt3a’s actions is that cocaine transiently induces IEG expression and this is
associated with an increase in dendritic spine density whereas Dnmt3a
overexpression reduces IEG expression and increases spine density. Clearly
there are many genes that influence dendritic spine formation and spine
elimination. Within the IEG gene family Arc and ΔFosB have both been
implicated in dendritic spine plasticity (Greer and Greenberg, 2008; Maze et al.,
2010). Increased Arc promotes internalization of AMPA receptors thereby
weakening synaptic strength and making synapses more prone to elimination,
whereas ΔFosB increases spine density through yet to be determined
mechanisms. Although IEGs are transiently increased by psychostimulants,
Freeman and colleagues have found that Arc and several other IEGs (but not
FosB) are persistently downregulated for up to 100 days after the last selfadministration session (Freeman et al., 2008). Such regulation fits perfectly with
cocaine induced upregulation of Dnmt3a and increased density of dendritic
spines at longer term timepoints.
106
CHAPTER 3 FIGURES
Figure 3-1
107
Fig. 3-1. Optimization of methylated DNA IP in brain tissue (a) mDIP
overview. DNA is purified, sonicated, and immunoprecipitated using an antibody
against methylated cytosine. After IP, samples can be analyzed via qPCR or
amplified for microarray studies. (b) DNA size distribution after increase numbers
of 10s sonication pulses. 4 pulses replicates the 300-1000bp size distribution
reported in other studies. (c) PCR analysis of known methylated (IAP, H19.1,
H19.3) and unmethylated (CSA, ActB) loci at varying IP conditions (antibody to
DNA ratios) and after whole genome amplification (WGA). As the concentration
of antibody is increased, the specificity for methylated DNA enrichment is lost. A
1:2 ratio exactly replicates finding from cell culture (90 fold enrichment of IAP, 30
fold enrichment of H19 loci). WGA under these conditions replicates these
findings as well. (d) mDIP (top panel) comparisons of differentially expressed
genes in striatum compared to olfactory bulb. Bottom panel displays relative
mRNA expression of each of these differentially expressed genes as reported in
the Allen Brain Atlas project. TH (tyrosine hydoxylase) and Drd1a (dopamine D1
receptor) are differentially methylated, with TH displaying generally high levels of
methylation (20 fold enrichment) in both samples.
108
Figure 3-2
109
Fig. 3-2. Cocaine-induced mDIP-Chip analysis (a) Venn diagrams comparing
significant mDIP genelist (1707 genes, p<0.001) to differentially expressed genes
after 1 and 24hrs (544 and 130 genes, fold change (FC) cutoff 1.3x, p<0.05) (b)
increasing significance cutoff to 1.5 fold dramatically reduces the number of
significantly methylated genes (61 genes, FC 1.5x, p<0.001) as well as the
number of overlapping differentially expressed transcripts. (c) Examples of
promoter plots from genes from (a) with individual CpG sites annotated as
vertical red lines. Red- enrichment with cocaine, blue- enrichment with saline,
black- cocaine-induced fold change in enrichment.
110
Figure 3-3
111
Fig. 3-3. Bisulfite sequencing example. Top panel is a promoter plot example
of differential methylation at the Htr3a gene. Green box around promoter
represents the amplicon that bisulfite sequencing primers were designed against.
Pink vertical lines within box represent individual CpG sites. Bar graph below is
quantification of % methylation.
112
Figure 3-4
113
Fig. 3-4. Intra-NAc Dnmt3a induced mRNA expression profiling (a) NAc
tissue punches were taken from HSV-GFP or HSV-Dnmt3a-GFP positive tissue
and mRNA overexpression of Dnmt3a relative to GFP was verified (b) global
DNA methylation analysis shows that Dnmt3a overexpression increases total
NAc DNA methylation levels. (c) mRNA expression profiling of 100 genes (see
table 3-2) was carried out. Overall, 18 genes were significantly regulated. 10 out
of 12 genes in the immediately early gene (IEG) family are significantly
downregulated. (d) mRNA expression of 8 other genes that were significantly
regulated by Dnmt3a overexpression.
114
Figure 3-5
115
Fig. 3-5. RG108 induced mRNA expression profiling. 100 μM RG108 in 0.2%
DMSO or 0.2% DMSO alone were applied to cultured E18 rat striatal neurons.
After 24 hours, half of the samples were stimulated under depolarizing conditions
(60 mM KCl) for 1 hour. RNA was then extracted and microarray analysis was
carried out. (a) heatmap of 95 genes significantly upregulated by RG108 (*, FC
>1.3x, p<0.05) and how these genes are regulated by KCl. More than 65% of
these genes are also upregulated by KCl (b) heatmap of 131 genes significantly
upregulated by KCl (*, FC >2x, p<0.05) and how these genes are regulated by
RG108. More than 40% of these genes are similarly regulated by RG108
(c)
Bar graphs of mRNA expression of Gadd45B. A gene that is significantly
regulated on both heatmaps, as indicated by a “ ” in (a) and (b).
116
Table 3-1. Candidate methylated genes
Top
hypermethylated
genes
Tph2
Syt4
Sst
Snca
Slc18a2
Scnm1
Rhoc
Pml
Pdgfrb
Olig3
Olfr196
Myc
Msh5
Lmna
Lim2
Lgals8
Lgals6
Lgals4
Lgals1
Lcat
Kcnu1
Kcnmb1
Kcnj6
Kcnh6
Htr4
Htr3a
Htr1d
Hdac4
Hbp1
Gadd45gip1
fosB
Chrm4
Cebpb
Casp6
Cart
Capg
Camkk1
Camk2a
Adrb3
Fold
methylation
1.36
1.38
1.29
1.39
1.28
1.27
1.27
1.26
1.28
1.68
2.46
1.80
1.72
1.27
1.92
1.38
1.38
1.38
1.31
1.84
1.45
2.18
1.44
1.28
1.29
1.29
1.33
1.41
1.90
1.68
1.31
1.31
1.26
1.38
1.28
1.30
1.31
1.30
1.27
Validated fold
mRNA
expression
bisulfite
sequencing
validation?
no
no
0.9 (24hr)
0.91 (24hr)
no
no
0.9 (24hr)
0.92 (24hr)
no
no
no
no
no
no
no
no
1.64 (1hr)
no
no
no
no
no
0.85 (24hr)
no
117
Top hypomethylated
Cask
Dicer1
Dlgh4 (psd-95)
Drd3
Ehmt2 (g9a)
Grin2d
Hdac7a
Nrxn2
Ntrk2
Pah
Rab36
Rorb
Strn4
Tktl1
> 1.5 fold cutoff
Also on 1hr or 24hr microarray
genelist
Implicated in cocaine reward
Fold
methylation
0.79
0.76
0.77
0.77
0.73
0.76
0.71
0.75
0.78
0.68
0.64
0.70
0.65
0.64
Validated
fold mRNA
expression
bisulfite
sequencing
validation?
no
0.75 24hr
no
no
no
no
no
no
1.15 24hr
no
118
Table 3-2. NAc mRNAs analyzed after overexpression of Dnmt3a
Gene family
Metabotropic glutamate receptors
Ionotrophic glutamate receptors
Dopamine receptors
Gaba-a receptors
serotonin receptors
GPCR's (other)
Ion channels
Spine structural genes
Actin regulatory genes
Galnectins (ex: Lgals1)
Neurotophins/neuropeptides
Cytokines (EX: Tnfa)
Kinases (ex: Camk2a)
miRNA processing (ex: Dicer1)
Glucocorticoid family
NFkB signaling
Creb family
Histones
Chromatin modifying enzymes
Immediate early genes
Other
total
# genes
analyzed
2
5
3
4
3
2
3
3
6
3
13
1
8
1
2
3
4
2
15
12
5
100
# genes
significantly
regulated
0
0
0
0
0
0
0
2
0
1
0
1
2
1
0
0
0
0
1
10
0
18
upregulation
downregulation
119
Suppl figure 3-1
Supplemental Figure 3-1. Comparison of DNA Methylation levels with
mRNA expression levels. Displayed here is the mRNA expression level (shown
as percentile) from a list of genes that lie in the top 20th percentile of enriched
methylated genes (~3900 genes). One can see that the majority of genes
promoters that have high methylation also have low mRNA expression.
120
CHAPTER 4
CONCLUSION
SUMMARY OF FINDINGS
DNA methylation and Behavior
Prior data on the behavioral role of histone deacetylases and repressive
histone methyltransferases have found that these histone modifying enzymes
attenuate the rewarding effects of cocaine (Kumar et al., 2005; Maze et al., 2010;
Renthal et al., 2007; Wang et al., 2010). If transient covalent histone
modifications play a role in addiction (a persistent illness), then what is the role of
more stable covalent modifications such as DNA methylation? In this thesis, I
present data demonstrating that Dnmt3a also reduces cocaine reward as
measured by conditioned place preference. Therefore, these data provide the
third line of evidence suggesting that mechanisms that promote global gene
repression may serve as a therapeutic treatment for addiction. Conversely, the
data from this thesis demonstrate the second line of direct evidence that blocking
NAc gene repressive mechanisms is antidepressant (the other evidence comes
from HDAC inhibitor studies by Covington et al., 2009).
Regulation of DNA methyltransferases
I found that Dnmt3a is persistently upregulated after prolonged cocaine
withdrawal (30 days) or chronic social defeat stress (10 days after the last
stressor). Contrary to what was expected, I also discovered that Dnmt3a is very
121
dynamically regulated (both up and down regulated) by the early effects of
cocaine. This type of biphasic mRNA regulation (Fig 2-1a) of a chromatin
modifying enzyme has never been found in the addiction field, nor has such
persistent long-term regulation been observed in the NAc (further detailed in
discussion, Chapter 2).
Gene targets
In the face of such complex regulation of Dnmt3a, what are the genomic
loci that are regulated by methylation? Carefully standardized whole genome
promoter analysis after chronic cocaine injections largely point to a lack of
cocaine-induced DNA methylation at gene promoter regions. Two limitations of
this experiment and its interpretation (also mentioned below) include the fact that
this is only one timepoint in an obviously complex system and that the region
analyzed (gene promoters) only assays ~1.5% of the mouse genome. In support
of these caveats, it is clear that DNA methylation regulates transcription of
specific genes. Using viral mediated overexpression and pharmacological
inhibition of DNA methylation I discovered that DNA methylation either directly or
indirectly regulates immediate early gene expression. Since immediate early
genes represent the first wave of transcriptional activation in response to cocaine
these data warrant more thorough DNA methylation analysis at various
timepoints and at several genomic loci in and around immediate early genes
(particularly at enhancer elements, as detailed in Chapter 3 Discussion).
122
DNA methylation and neural plasticity
In the addiction field, the most well documented persistent neuronal
adaptation is cocaine’s ability to increase dendritic spine density in the NAc.
Therefore, does DNA methylation influence dendritic spine density? Viralmediated Dnmt3a overexpression was found to be sufficient to increase dendritic
spine density. Moreover, NAc specific pharmacological blockade of DNA
methylation demonstrated that methylation is also necessary for cocaine induced
increases in spine density. Finally, detailed spine morphology analysis brought a
new understanding to the specific types of dendritic spines regulated by cocaine.
I found that thin spines—which are thought to represent newly formed, immature
spines—are the major subtype of spines regulated by cocaine and importantly,
by DNA methylation.
DNA METHYLATION SPECIFIC FUTURE DIRECTIONS
The role of Gadd45β in addiction
As mentioned in the Chapter 1, Gadd45β is a putative DNA demethylase
(Ooi and Bestor, 2008). Data in my thesis work have linked this gene to being
regulated by cocaine as well as regulated by DNA methylation. Specifically, I
have found that expression of this gene is reduced by Dnmt3a overexpression
and increased by Dnmt inhibition. Adding to this complexity, I see that Gadd45β
is also biphasically regulated by cocaine, with upregulation at 1 hour and
123
downregulation at 4 hours after a cocaine injection. Of note, this regulatory
pattern precedes and inversely correlates with Dnmt3a’s expression. Taken
together, these data further suggest that cocaine induced DNA methylation is
highly dynamic, and opens the possibility of cross-talk between Dnmt3a and
Gadd45β. In light of these data, I have obtained both RNAi and overexpression
constructs (generous gift Dr. H. Sun, Ma et al., 2009) and it will hence be
important to test how manipulating Gadd45β influences cocaine induced dendritic
spines and behavior.
Identifying cocaine-induced DNA methylation
As suggested above, I think the best strategy to identifying cocaineinduced methylation is a hypothesis driven approach. This includes careful
timecourse and detailed genomic location analysis of the methylation status of
immediate early genes such as Gadd45β, FosB, c-Fos, and Arc. In light of recent
ChIP sequencing data (Kim et al., 2010), the most likely region of regulation of
DNA methylation is in highly conserved enhancer elements. Since the gold
standard for methylation analysis, bisifulte sequencing, cannot differentiate
between
cytosine
methylation
and
the
newly
discovered
brain
hydroxymethylation, I do not think it is worthwhile to analyze DNA methylation via
bisulfite sequencing, unless proven otherwise. The most convincing approach
would be to perform both mDIP experiments in addition to chIP experiments with
a methyl-domain binding protein, such as MeCP2. Since I have also found that
FosB, c-Fos, and Arc (Gadd45β not assessed) have long-term (1 month
124
withdrawal) alterations in gene inducibility, it will also be very interesting to look
at DNA methylation after long periods of withdrawal at these gene enhancers.
Role of DNA methylation in more long term behaviors
Now that the baseline manipulations of DNA methylation have been
performed, the next key step will be to characterize how manipulating DNA
methylation after cocaine experience influences cocaine craving and relapse
behavior. Importantly, a collaboration with a self-administration lab has already
been set up and experiments are underway!
GENERAL FUTURE DIRECTIONS
Regulation of chromatin structure has greatly advanced our basic
understanding of the pathophysiology of the addicted and depressed state, and
offers a fundamentally new approach for the development of more effective
treatments for drug addiction and depression. Behavioral studies to date have left
open major avenues of investigation as well as leaving key mechanistic
questions unanswered. The hypothesis that ‘blocking cocaine’s ability to create a
more permissive chromatin state serves as a potential therapeutic treatment for
addiction’ needs further investigation. A promising line of research would be to
assess the behavioral function of enzymes that mediate transcriptional activation
such as H3K4, H3K36, or H3K79 methyltransferases. To date, the majority of
studies have focused on the enzymes that mediate transcriptional repression—
such as HDACs, H3K9 methyltransferases, and DNA methyltransferases.
125
Moreover, despite having similar behavioral outcomes, the gene targets of
HDACs, G9a, or DNMT inhibitors remain unknown. It is not known if these
manipulations are affecting primarily cocaine-expressed genes (primed or
desensitized genes) (Renthal et al., 2007) or completely novel genes—as was
found in a recent study comparing target genes affected in the NAc by the HDAC
inhibitor MS-275 or by an antidepressant medication (Covington et al., 2009).
Furthermore, the degree of cross-talk and gene target overlap of each
manipulation is not known. For example, since class III HDACs (sirtuins) regulate
excitability of NAc neurons (Renthal et al., 2009), does this extend to other
chromatin modifying enzymes such as DNA methyltransferases? Similarly, do
manipulations that inhibit DNA methylation also affect histone acetylation, and
vice versa, as has been shown in the learning and memory field (Miller et al.,
2008)?
Similar to behavior manipulations, a more thorough analysis of cocaine’s
effects on chromatin structure and function demand attention. Two key
weaknesses of the published genome-wide ChIP-chip study are that: 1) the
immunoprecipitation utilized antibodies that were not specific to individual PTMs,
and 2) analysis was localized only to gene promoter regions (an inherent
limitation of ChIP-chip). For the next step in gaining insight into why, for example,
cocaine globally reduces H3K9me2, it will be crucial to immunoprecipitate with a
specific anti-H3K9me2 antibody in a completely unbiased genome-wide setting
using ChIP-seq (ChIP-sequencing) technology.
126
In addition to more thorough genomic analysis and more thorough
characterization of novel modifications, two crucial technical challenges exist in
linking chromatin function with gene expression. First is differentiating causality
from correlation. At best, the presence of a given PTM at a given gene promoter
can only be associated with a gene’s expression. Such correlation is even further
confounded in examples such as the CART gene promoter, which has a
bidirectional methylated H3 profile (see Fig. 1-1B). Even with pharmacological or
overexpression
studies,
one
cannot
conclusively
determine
whether
overexpression of Dnmt3a, for example, directly vs. indirectly influences
transcription of the CART gene because such overexpression has global effects.
To address this question, one must specifically target DNA methylation to the
CART gene promoter. One possible route of accomplishing this very challenging
task could be fusing a chromatin modifying enzyme to a protein that specifically
binds to the CART gene promoter. This could be accomplished by knocking in
specific ‘targetable’ sequences at a promoter, or perhaps by designing zinc-finger
peptides to target specific sequences of DNA. This approach holds promise since
it has proven successful in cell culture using a DNA methyltransferase fused to a
zinc finger protein (Smith et al., 2008). If successfully applied in vivo, this could
prove to be an extremely powerful tool in causally establishing the functional
influence on transcription as well as providing insight in the in vivo half-life of a
given PTM.
A second major technical challenge lies in further refining cell specific
tools to analyze chromatin state within a given brain region of interest like the
127
NAc. The importance of this issue has overtones throughout this thesis. In the
second chapter, I found that Dnmt3a is subtly (but significantly) regulated by
cocaine and stress (Fig 2-1a) and, in the third chapter, I was ultimately unable to
detect large differences in mRNA or any differences in methylated DNA
expression in response to chronic cocaine (Fig. 3-2a). Here, I am left with
wondering if perhaps bona fide regulation could be found in purified types of
neurons. Because brain tissue is highly heterogeneous and contains many
different classes of neurons and glia, data generated from ChIP and mRNA
studies may be a poor reflection of what is actually occurring in specific neurons.
Furthermore, because these analyses are carried out on tissue homogenates, it
is difficult to ascertain whether differences reflect large changes that occur in
absolute levels in a minority of cells, such as neuronal ensembles, or incremental
changes that are occurring in a majority of cells in that region. Again referring to
the CART promoter example (Fig. 1B), could this bidirectional profile be due to
differences occurring in different populations of cells? This scenario is entirely
plausible since NAc neurons which express Gs-coupled dopamine D1 receptors
have vastly different baseline and cocaine-induced mRNA expression profiles
compared to Gi-coupled D2 receptor containing neurons (Heiman et al., 2008;
Lobo et al., 2006). Theoretically, since ChIP has been successfully combined
with FACS (Flourescence Activated Cell Sorting) of brain tissue (by sorting
NeuN+ cells to isolate neurons specifically), it should be possible to ChIP even
further purified populations of cells (Jiang et al., 2008).
128
Altogether, such studies of chromatin open a new window on the
molecular basis of drug addiction and depression. The hope is that as novel
insight is achieved, it will be possible one day to take advantage of this
information to develop better diagnostic tests for addiction and depression and to
develop improved treatments and ultimately preventive measures.
129
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